Vitamin functionalized gel-forming block copolymers for biomedical applications

ABSTRACT

Gel-forming block copolymers were prepared comprising i) a central hydrophilic block consisting essentially of a divalent poly(ethylene oxide) chain and ii) two peripheral monocarbonate or polycarbonate hydrophobic blocks linked to the central block by linking groups bearing one or more hydrogen bond forming *—N(H)—* groups. The hydrophobic blocks comprise one or more vitamin-bearing subunits. The gel-forming block copolymers can be used to prepare various biodegradable and/or biocompatible hydrogel and organogel drug compositions, in particular antimicrobial and/or anti-tumor drug compositions. The hydrogel compositions have utility in depot injections for drug delivery. The hydrogen bonding *—N(H)—* group(s) provide longer in vivo lifetime of the hydrogel before degradation and a more prolonged and controlled release rate of a hydrophobic drug compared to similar hydrogels prepared from poly(ethylene glycol).

PARTIES TO A JOINT RESEARCH AGREEMENT

This invention was made under a joint research agreement betweenInternational Business Machines Corporation and the Agency For Science,Technology and Research.

BACKGROUND

Hydrogels and organogels produced from the self-assembly of syntheticpolymers have an inexhaustible potential to serve as a delivery matrixfor a vast range of pharmaceutical, cosmetic and dietary products.Recent developments in polymer chemistry have enabled polymers to besynthesized with well-controlled composition and architecture. Highlyversatile orthogonal functionalization strategies also allow gelation ofsuch polymers and containment of drug payload through one or acombination of the following association mechanisms: hydrophobicinteractions, ionic interactions, hydrogen bonding, physicalentanglement of macromolecules and chemical crosslinking of the matrix.

A number of physical gel systems have been formulated using the‘ABA’-type triblock copolymers, and the polymeric amphiphiles can bedesigned with either the ‘A’ or ‘B’ constituent blocks to be hydrophilicor hydrophobic. Many of such systems engage the use of poly(ethyleneglycol) (PEG) as the uncharged hydrophilic constituent block for itsbiocompatibility and non-toxicity. As for hydrophobic portion(s), someof commonly selected blocks are poly(L-lactide) (PLLA), poly(D-lactide)(PDLA), poly(glycolide) (PGA), and poly(caprolactone) (PCL), which canbe prepared either as the middle ‘B’ block (e.g., PEG-b-PGA-b-PEG) or asthe terminus ‘A’ blocks (e.g., PLLA-b-PEG-b-PLLA). Aqueous mixture ofenantiomeric triblock copolymers of the PLLA and PDLA-containingpolymers could also give rise to physical gels formed viastereocomplexation.

Polymeric gel systems can be broadly classified as organogels orhydrogels, depending on the dispersion media used. Organogels have anorganic liquid phase that is immobilized by a 3D physically crosslinkednetwork of intertwined fibers of self-assembled polymer chains. Variouskinds of organic material can be used to make up the liquid phase (e.g.,organic solvents, mineral oil, plant oil, and combinations of theforegoing). Organogels are mainly used for cosmetics/dietaryapplications while relatively much fewer organogels are being evaluatedas drug/vaccine delivery matrices. This is primarily due to the scarceamount of information available regarding the toxicology andbiocompatibility of the gel-forming polymers and their degradedproducts. Nevertheless, when toxicity concerns are circumvented,organogels have potential for use as topical formulations owing to theenhanced dermal permeation capacities of typical organogels. Alternativemodes of application include oral and trans-mucosal as well assubcutaneous depot injections. Herein, a depot is a body area in which asubstance (e.g., a drug) can be accumulated, deposited, or stored andfrom which it can be distributed. A depot injection is an injection of asubstance in a form that tends to keep it at or near the site ofinjection so that absorption occurs over a prolonged period.

Hydrogels, which are prepared in water, are more widely-studied comparedto organogel systems. Most of the commonly used hydrogel-forming‘ABA’-type triblock copolymers (e.g., PLLA-b-PEG-b-PLLA andPCL-b-PEG-b-PCL) require high polymer concentration and hydrophobiccontent for hydrogel formation. For instance, (PLLA-b-PEG-b-PLLA)containing lactide content of 17 wt. % to 37 wt. % requires a minimumconcentration of about 16 wt. % of the triblock copolymer for gelation.Such a high proportion of hydrophobic constituents could give rise toadverse physiological effects during in vivo degradation. Thus, it isdesirable to develop polymeric materials that can form hydrogels at alow concentration.

According to a 2008 World Health Organization (WHO) survey, breastcancer comprises of 22.9% of all cancers (excluding non-melanoma skincancer) and its mortality rate is around 13.7% worldwide. In Europe, theincident rate of breast cancer is even higher, reaching 28%. Treatmentof breast cancer may vary according to the size, stage and rate ofgrowth, as well as the type of tumor. There are currently three maincategories of adjuvant, or post-surgery, therapy. These includehormone-blocking therapy, chemotherapy and monoclonal antibodies (mAbs)therapy. The latter involves the utilization of mAbs to target specificcells or proteins towards the treatment of disease by inducing,enhancing, or suppressing an immune response. It can be used inconjunction with either hormone-blocking therapy or chemotherapy toenhance the efficacy of cancer treatment.

Studies have shown that the human epidermal growth factor receptor 2(HER2) genes are amplified and/or overexpressed in 20% to 25% ofinvasive breast cancers. These HER2-positive breast cancers havesignificantly lower survival rates compared to HER2-negative breasttumors. The HER2-positive breast tumors are most likely to showunrestrained growth and division of cells, thus increasing the incidenceof cancer development. Herceptin is a recombinant humanized mAb that canselectively bind to HER2 proteins, thereby regulating the otherwiseuncontrollable cancer cell growth. It is also a US Food and DrugAdministration (FDA)-approved therapeutic for the treatment ofHER2-positive breast cancer. Intravenous administration is the currentmode of herceptin delivery in most clinics. However, many controversiessurround the optimal mode of delivery in terms of duration and dosage.Recently, F Hoffmann-La Roche reported a phase 3 clinical trial (HannaHstudy) involving the subcutaneous (versus intravenous) administration of(neo)adjuvant herceptin in patients with HER2-positive breast cancer.The formulation contained a fixed dose of herceptin and recombinanthuman hyaluronidase (rHuPH-20), a class of enzymes that temporarilydegrades interstitial hyaluronan in the subcutaneous space, as anexcipient. The study found that therapeutic efficacy of subcutaneousdelivery of herceptin was comparable to the traditional intravenousroute but the therapy had the advantage of improved patient convenience,better compliance, reduced pharmacy preparation times, and optimizationof medical resources.

The foregoing illustrates that an ongoing need exists for moreefficacious drug and antibiotic formulations. More specifically,formulations are needed for improved efficacy of subcutaneous treatmentsused in cancer therapy.

SUMMARY

Accordingly, a block copolymer is disclosed of formula (1-C):

wherein

atomic centers numbered 1 to 6 of each carbonate unit within parenthesesare backbone atomic centers of the block copolymer,

d′ is a positive number having an average value of 100 to 600,

each m′ is an independent positive number having an average valuegreater than 0 and less than or equal to 10,

each L^(d) is independently a single bond or a divalent linking groupcomprising 1 to about 15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons.

Also disclosed is a drug composition, comprising:

about 4 wt. % to about 10 wt. % of an above-described gel-forming blockcopolymer;

a solvent; and

about 0.0001 wt. % to about 10 wt. % of a drug;

wherein

weight percent (wt. %) is based on total weight of the drug composition,and

the drug composition is a gel wherein the block copolymer, the solvent,and the drug are associated by noncovalent interactions.

Further disclosed is a method of treating a cancer, comprisingperforming an in vivo depot injection of the above-described drugcomposition near or in contact with a cancerous tumor.

Also disclosed is a method of preparing the above-described blockcopolymer, comprising heating under vacuum with agitation at atemperature of about 50° C. to about 150° C. a mixture consisting of i)a cyclic carbonate monomer comprising a pendent group containing acovalently bound form of a vitamin and ii) a dinucleophilic polymericinitiator for a ring opening polymerization (ROP) of the cycliccarbonate monomer, the polymeric initiator comprising a poly(ethyleneoxide) chain and two terminal *—NH₂ groups, thereby forming the blockcopolymer.

The above-described and other features and advantages of the presentinvention will be appreciated and understood by those skilled in the artfrom the following detailed description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a graph showing the storage (G′) and loss (G″) moduli ofblank hydrogels containing 4 wt. % and 8 wt. %VitE1.25-PEG(20k)-VitE1.25 in HPLC water (Example 15 and Example 16,respectively). Herein, weight percent (wt. %) is based on total weightof the hydrogel unless otherwise indicated.

FIG. 1B is a graph showing the storage (G′) and loss (G″) moduli ofblank hydrogels containing 4 wt. % and 8 wt. % VitE2.5-PEG(20k)-VitE2.5in HPLC water (Example 17 and Example 18, respectively).

FIG. 1C is a graph showing the viscosity dependence on shear rate ofblank hydrogels containing 4 wt. % and 8 wt. %VitE1.25-PEG(20k)-VitE1.25 in HPLC water (Example 15 and Example 16,respectively).

FIG. 1D is a graph showing the viscosity dependence on shear rate ofblank hydrogels containing 4 wt. % and 8 wt. % VitE2.5-PEG(20k)-VitE2.5in HPLC water (Example 17 and Example 18, respectively).

FIG. 1E is a graph showing viscosity dependence on shear rate of blankorganogels containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5 and 10 wt. %VitE8.5-PEG(20k)-VitE8.5 organogels in KOLLIPHOR RH40 (Example 19 andExample 20, respectively).

FIG. 2 is a graph showing the dynamic step strain amplitude test (y=0.2or 100%) of blank hydrogel Example 15 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25, and a herceptin-loaded hydrogel Example 24containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1 wt. % herceptin.

FIGS. 3A and 3B are scanning electron micrographs (SEM) images ofcryo-fixed blank hydrogels containing 4 wt. % (Example 15) and 8 wt. %(Example 16) VitE1.25-(PEG20k)-VitE1.25.

FIGS. 3C and 3D are scanning electron micrographs (SEM) images ofcryo-fixed blank hydrogels containing 4 wt. % (Example 17) and 8 wt. %(Example 18) VitE2.5-(PEG20k)-VitE2.5.

FIG. 4A is a graph showing the release rate of sodium nicotinate fromloaded hydrogels:

a) Example 21 containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt.% sodium nicotinate (diamonds), and

b) Example 22 containing 8 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt.% sodium nicotinate (circles), and

c) Example 23 containing 4 wt. % VitE2.5-PEG(20k)-VitE2.5 and 0.3 wt. %sodium nicotinate (triangles).

FIG. 4B is a graph showing the release rate of herceptin from loadedhydrogels:

a) Example 24 containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt.% herceptin and

b) Example 25 containing 4 wt. % VitE2.5-PEG(20k)-VitE2.5 and 1.0 wt. %herceptin.

FIG. 4C is a graph showing the release rate of doxycycline (DXY) fromorganogels:

a) Example 26 containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5 and 1.0 wt. %doxycycline, with and without added lipase, and

b) Example 27 containing 10 wt. % VitE8.5-PEG(20k)-VitE8.5 and 1.0 wt. %doxycycline, with and without added lipase.

FIG. 5 is a bar graph showing the percentage of viable human dermalfibroblast (HDF) cells after treating the cells with blank hydrogelExamples 15 and 16 (containing 4 wt. % and 8 wt. %VitE1.25-PEG(20k)-VitE1.25, respectively, grey bars) and blank hydrogelExamples 17 and 18 (containing 4 wt. % and 8 wt. %VitE2.5-PEG(20k)-VitE2.5, black bars).

FIG. 6 is a bar graph showing the percentage of viable human dermalfibroblast (HDF) cells after treating the cells with blank organogelExample 19 (containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5), doxycyclineloaded hydrogel Example 26 (containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5and 1 wt. % doxycycline), blank organogel Example 20 (containing 10 wt.% VitE8.5-PEG(20k)-VitE8.5), and doxycycline loaded hydrogel Example 27(containing 10 wt. % VitE8.5-PEG(20k)-VitE8.5 and 1 wt. % doxycycline).

FIG. 7 is a bar graph showing the percentage of viableHER2/neu-overexpressing human breast cancer BT474 cells as a function ofherceptin concentration after treating the cells with:

(a) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 48 hours,

(b) herceptin solution for 48 hours,

(c) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 120 hours, and

(d) herceptin solution for 120 hours, each performed using a herceptinconcentration of 0.0005 wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1wt. %, and 0.5 wt. %. The herceptin loaded hydrogels were prepared usingthe procedure of Example 24.

FIG. 8 is a bar graph showing the viability of MCF7 cells as a functionof herceptin concentration when treated with:

(a) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 48 hours,

(b) herceptin solution for 48 hours,

(c) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 120 hours, and

(d) herceptin solution for 120 hours, each performed using a herceptinconcentration of 0.0005 wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1wt. %, and 0.5 wt. %. The herceptin loaded hydrogels were prepared usingthe procedure of Example 24.

FIG. 9 is a bar graph showing the viability of human dermal fibroblast(HDF) cells as a function of herceptin concentration when treated with:

(a) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 48 hours,

(b) herceptin solution for 48 hours,

(c) herceptin loaded hydrogel containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 for 120 hours, and

(d) herceptin solution for 120 hours, each performed using a herceptinconcentration of 0.0005 wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1wt. %, and 0.5 wt. %. The herceptin loaded hydrogels were prepared usingthe procedure of Example 24.

FIG. 10 is a series of optical photomicrographs of CD45 andhaematoxylin/eosin (H&E) stained mice tissue after treatment with blankhydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25), retrieved at1, 2, 4 and 6 weeks post injection. Arrows indicate the region wherehydrogel is present. At week 6, the hydrogel has mostly degraded andcould not be distinctly identified. The scale bar represents 200micrometers.

FIG. 11 is a series of mouse drawings showing the biodistribution ofALEXA FLUOR 790-labeled herceptin within BT474-tumor bearing mice over a13 day period. The herceptin was delivered once to the mice in threeways: i) using herceptin loaded hydrogel (Example 24) deliveredsubcutaneously (“Herceptin-loaded hydrogel, S.C.”), ii) herceptinsolution delivered intravenously (“Herceptin solution, I.V.”), and iii)herceptin solution delivered subcutaneously (“Herceptin solution,S.C.”). On Day 13, the mice were sacrificed and organs involved in drugclearance and metabolism as well as tumor tissue were excised andimaged. From left: liver, spleen, lungs, kidneys, tumor.

FIG. 12A is a graph showing the change in body weight of BT474-tumorbearing mice after one injection using various herceptin formulations,including blank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25)delivered subcutaneously (“Blank Gel”) herceptin solution deliveredintravenously (“Herceptin Sol (IV, 30 mg/kg, Once”)), herceptin solutiondelivered subcutaneously (“Herceptin Sol (SC, 30 mg/kg, Once”)), andherceptin loaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25and 1.0 wt. % herceptin) delivered subcutaneously (“Herceptin Gel (SC,30 mg/kg, Once”)). The herceptin dosage was 30 mg/kg.

FIG. 12B is a graph showing change in tumor size of BT474-tumor bearingmice after one injection using various herceptin formulations, includingblank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25) deliveredsubcutaneously (“Blank Gel”) herceptin solution delivered intravenously(“Herceptin Sol (IV, 30 mg/kg, Once”)), herceptin solution deliveredsubcutaneously (“Herceptin Sol (SC, 30 mg/kg, Once”)), and herceptinloaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0wt. % herceptin) delivered subcutaneously (“Herceptin Gel (SC, 30 mg/kg,Once”)). The herceptin dosage was 30 mg/kg.

FIG. 13 is a series of photomicrographs showing tumor cells ofBT474-tumor bearing mice after terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) staining at 28 days after one injectionusing various herceptin formulations, including blank hydrogel Example15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25) delivered subcutaneously (“BlankGel (S.C.)”), herceptin solution delivered intravenously (“Her Sol(I.V.)”), herceptin solution delivered subcutaneously (“Her Sol(S.C.”)), and herceptin loaded hydrogel Example 24 (4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. % herceptin) deliveredsubcutaneously (“Her Gel (S.C.”)). The herceptin dosage was 30 mg/kg.The scale bar represents 100 micrometers. The tumor cells treated withherceptin, regardless of the formulation used, were mostly apoptotic,indicating that anti-tumor mechanism was based on herceptin-inducedapoptosis.

FIGS. 14A and 14B are graphs showing the changes in tumor size (FIG.14A) and body weight (FIG. 14B) of BT474-tumor bearing mice after fourweekly administrations of herceptin solution delivered intravenously(“Herceptin Sol (IV, 4×10 mg/kg, Weekly”) and subcutaneously (“HerceptinSol (IV, 4×10 mg/kg, Weekly”) compared to herceptin loaded hydrogel(Example 24) delivered once subcutaneously (“Herceptin Gel (SC, 40mg/kg, Once”). The total herceptin dosage was 40 mg/kg in each group.

FIG. 15 is a series of photomicrographs showing tumor cells ofBT474-tumor bearing mice after TUNEL staining at 28 days followinginjection of herceptin solution and herceptin loaded hydrogel asdescribed above for FIGS. 14A and 14B. Herceptin solution deliveredintravenously is labeled “Her Sol (I.V., weekly”). Herceptin solutiondelivered subcutaneously is labeled “Her Sol (S.C., weekly”). Herceptinloaded hydrogel (Example 24) delivered subcutaneously is labeled “HerGel (S.C., one-time”). Herceptin solution injections were performed on aweekly basis while herceptin loaded hydrogel (SC) was injected once onthe first day of treatment. The total dosage was 40 mg/kg. The scale barrepresents 100 micrometers.

FIG. 16 is a series of photomicrographs of mice heart, lung, liver andkidney cells after H&E staining at 28 days post injection of herceptinsolution formulations (intravenous and subcutaneous) performed on aweekly basis and herceptin loaded hydrogel (Example 24) injectedsubcutaneously once on the first day of treatment, as described abovefor FIG. 15. The scale bar represents 100 micrometers.

FIG. 17A is a bar graph of G′ values of cationic polymer loadedhydrogels Example 28 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and0.1 wt. % cationic polymer VE/BnCl(1:30) in HPLC water) and Example 29(containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.1 wt. % cationicpolymer VE/PrBr(1:30) in HPLC water). Also shown is blank hydrogelExample 15 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25).

FIG. 17B is a graph of viscosity versus shear rate profile of thecationic loaded hydrogels of FIG. 17A.

FIG. 18A to 18C are bar graphs showing the killing efficiency againstStaphylococcus aureus (S. aureus), Escherichia coli (E. coli), andCandida albicans (C. albicans), respectively, of cationic polymer loadedhydrogels containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and variousconcentrations of VE/BnCl(1:30) or VE/PrBr(1:30). The concentration ofpolymer on the horizontal axis refers to cationic polymer.

FIG. 19 is a bar graph showing the number of viable bacterialcolony-forming units (CFU) after 18 hour treatment of E. coli. with:

a) blank organogel Examples 19 (“6.5VE-PEG20k-6.5VE”),

b) blank organogel Example 20 (“8.5VE-PEG20k-8.5VE”)

c) doxycycline loaded organogel Example 26 (“6.5VE-PEG20k-6.5VE 1 wt. %DXY”), and

d) doxycycline loaded organogel Example 27 “8.5VE-PEG20k-8.5VE 1 wt. %DXY”).

FIG. 20 is a bar graph showing the killing efficiencies of threesolutions against C. albicans:

a) VE/PrBr(1:15) alone at 1.0MIC (250 ppm) against C. Albicans,

b) VE/PrBr(1:15)/fluconazole solution prepared with VE/PrBr(1:15) at0.5MIC (125 ppm) and fluconazole (2.5 ppm), and

c) fluconazole alone (5.0 ppm). MIC refers to minimum inhibitoryconcentration of the cationic polymer.

FIG. 21 is an isobologram demonstrating the synergy of theVE/PrBr(1:15)/fluconazole combination compared to VE/PrBr(1:15) aloneand fluconazole alone delivered by solution against C. albicans. Thesynergy is indicated by the drug combination dose that lies to the leftof the line of additivity, shown as a square inside the triangle.

FIG. 22 is a bar graph comparing the killing efficiency against C.albicans of:

(a) fluconazole loaded hydrogel Example 30 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.05 wt. % fluconazole) used at a loadedhydrogel concentration of 500 mg/L,

(b) cationic polymer/fluconazole loaded hydrogel Example 31 (containing4 wt. % VitE1.25-PEG(20k)-VitE1.25), 0.0156 wt. % cationic polymerVE/BnCl(1:30), and 0.001 wt. % fluconazole) used at a concentration offluconazole=10 mg/L and VE/BnCl(1:30)=156 mg/L (0.5MBC), and

(c) cationic polymer/fluconazole loaded hydrogel Example 32 (containing4 wt. % VitE1.25-PEG(20k)-VitE1.25), 0.0078 wt. % cationic polymerVE/BnCl(1:30), and 0.004 wt. % fluconazole) used at a concentration offluconazole=40 mg/L and VE/BnCl(1:30)=78 mg/L (0.25MBC). MBC refers tothe minimum bactericidal concentration.

FIG. 23 is a graph showing the release rate of fluconazole fromfluconazole loaded hydrogel Example 43 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.05 wt. % fluconazole (upper curve), andcationic polymer/fluconazole loaded hydrogel Example 44 containing 4 wt.% VitE1.25-PEG(20k)-VitE1.25), 0.3 wt. % cationic polymer VE/BnCl(1:30),and 0.3 wt. % fluconazole (lower curve).

FIG. 24 is an isobologram demonstrating the synergy of the combinationof VE/BnCl(1:30) and fluconazole for minimum bactericidal activity whendelivered by hydrogel. Synergy between the cationic polymer andfluconazole is shown by the drug combination dosage lying to the left ofthe line of additivity for each loaded hydrogel, represented by a squareinside the triangle. The upper square corresponds to Example 31containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25), 0.0156 wt. % cationicpolymer VE/BnCl(1:30), and 0.001 wt. % fluconazole. The bottom squarecorresponds to Example 32 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25), 0.0078 wt. % cationic polymerVE/BnCl(1:30), and 0.004 wt. % fluconazole.

FIG. 25 is a bar graph show the killing efficiency of a cationicpolymer/doxycycline (DXY) loaded hydrogel against Pseudomonas aeruginosa(P. aeruginosa). Four loaded hydrogels were prepared withVitE1.25-PEG(20k)-VitE1.25, DXY, and cationic polymer VE/BnCl(1:30)having DXY/VE/BnCl(1:30) ratios of 2.5 ppm/15.6 ppm (Example 33), 5ppm/15.6 ppm (Example 34), 1 ppm/31.2 ppm (Example 35), and 2.5 ppm/31.2ppm (Example 36), respectively. The killing efficiency was 100% at usingDXY loadings of 1 ppm to 5 ppm DXY and VE/BnCl(1:30) loadings of 15 ppmto 31 ppm.

FIG. 26 is an isobologram demonstrating the synergy of cationic polymerVE/BnCl(1:30) and doxycycline (DXY) against P. aeruginosa when deliveredby loaded hydrogels Example 33 and Example 35, indicated by acombination dosage to the left of the line of additivity, represented bya square inside the triangle. The left square corresponds to Example 35,the right square to Example 33. FIG. 26 shows the drug combination iseffective at extremely low doxycycline concentration.

FIGS. 27A and 27B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of S. aureus biofilms by:

a) blank hydrogel Example 15 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25,

b) cationic polymer loaded hydrogel Example 37 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30), and

c) cationic polymer loaded hydrogel Example 38 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30). Cationicpolymer VE/BnCl(1:30) and cationic polymer VE/PrBr(1:30) were loaded atminimum bactericidal concentrations (MBC) against S. aureus intoVitE1.25-PEG(20k)-VitE1.25 (4 wt. %) hydrogels.

FIGS. 28A and 28B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of E. coli biofilms by:

a) blank hydrogel Example 15 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25,

b) cationic polymer loaded hydrogel Example 37 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30), and

c) cationic polymer loaded hydrogel Example 38 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30). Cationicpolymer VE/BnCl(1:30) and cationic polymer VE/PrBr(1:30) were loaded atminimum bactericidal concentrations (MBC) against E. coli intoVitE1.25-PEG(20k)-VitE1.25 (4 wt. %) hydrogels.

FIGS. 29A and 29B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of C. albicans biofilms by:

a) blank hydrogel Example 15 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25,

b) cationic polymer loaded hydrogel Example 37 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30), and

c) cationic polymer loaded hydrogel Example 38 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30). Cationicpolymer VE/BnCl(1:30) and cationic polymer VE/PrBr(1:30) were loaded atminimum bactericidal concentrations (MBC) against C. albicans intoVitE1.25-PEG(20k)-VitE1.25 (4 wt. %) hydrogels.

FIG. 30 is a series of SEM images of S. aureus, E. coli, and C. albicansbiofilms after treatment with:

a) blank hydrogel Example 15 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25,

b) cationic polymer loaded hydrogel Example 37 containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30), and

c) Example 38 containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.0625wt. % VE/PrBr(1:30).

FIG. 31 is a graph showing the gel permeation chromatography (GPC)diagrams for Vit1.05-PEG10DA-VitE1.05 (Example 45) andVitE1.8-PEG20DA-VitE1.8 (Example 46).

FIG. 32 is a proton NMR spectrum of Example 45,Vit1.05-PEG10DA-VitE1.05, in CDCl3.

FIG. 33 is a graph showing the shear-thinning behaviour of hydrogelsformed with Example 45.

FIG. 34 is a graph showing the release rate of Herceptin from a 4 wt. %hydrogel formed with Example 46, Vit1.8-PEG20DA-VitE1.8, over a 45-dayperiod.

FIG. 35 is a set of photographs and staining images to evaluate thepresence of the hydrogel prepared with Example 46 in mouse tissue atvarious times during a 6 week period.

DETAILED DESCRIPTION

Disclosed are medical compositions comprising a mixture of ahydrogel-forming (“gel-forming”) block copolymer and a therapeutic agent(e.g., drug), which are in non-covalent association. The block copolymeris a linear polymer having a ABA type block structure (two peripheralblocks A and a central block B). The end units of the centralhydrophilic block B are covalently linked to respective end units of theperipheral hydrophobic blocks A by way of respective independentdivalent linking groups designated L′ and L″. At least one of L′ and L″contains an *—N(H)—* group capable of donating a hydrogen and/or anelectron pair to form a hydrogen bond. The gel-forming block copolymershave utility in medical compositions used in depot injections forcontrolled drug release. The hydrogen-bonding *—N(H)—* group(s) extendsthe in vivo gel lifetime before enzymatic degradation and provides alonger, more controlled rate of drug release compared to an otherwiseidentical block copolymer lacking the hydrogen-bonding *—N(H)—*group(s). The hydrophilic block B is a divalent poly(ethylene oxide)chain having a degree of polymerization (DP) of about 100 to about 600.Each of the hydrophobic blocks A can be a monocarbonate or apolycarbonate comprising 1 to about 10 carbonate subunits. The carbonatesubunits comprise a covalently bound form of a vitamin and are alsoreferred to herein as vitamin-bearing subunits. The vitamin-bearingsubunits comprise a carbonate backbone portion and a sidechain portionlinked to the backbone portion. The sidechain portion bears a covalentlybound form of a vitamin. The end groups of the block copolymer areselected from the group consisting of hydrogen and C₁-C₁₅ groups.

The hydrophobic blocks can comprise an optional second carbonatesubunit, which can act as a diluent for the vitamin-bearing subunitsand/or provide additional functionality (e.g., charge-containing amineor carboxylic acid groups) for enhancing the payload-carrying capacityand/or bioactive properties of the gel-forming block copolymers. In apreferred embodiment, the gel-forming block copolymers are non-charged.In another embodiment, each of the hydrophobic blocks consistsessentially of 1 to about 4 vitamin-bearing subunits, and each of theend groups is hydrogen.

The gel-forming block copolymer and the therapeutic agent (e.g.,antimicrobial cationic polymer, described further below) can bebiodegradable. The term “biodegradable” is defined by the AmericanSociety for Testing and Materials as degradation caused by biologicalactivity, especially by enzymatic action, leading to a significantchange in the chemical structure of the material. For purposes herein, amaterial is “biodegradable” if it undergoes 60% biodegradation within180 days in accordance with ASTM D6400. Herein, a material is“enzymatically biodegradable” if the material can be degraded (e.g.,depolymerized) by a reaction catalyzed by an enzyme.

The gel-forming block copolymer and therapeutic agent can bebiocompatible. A “biocompatible” material is defined herein as amaterial capable of performing with an appropriate host response in aspecific application.

The gel-forming block copolymer can form a gel in a solvent at atemperature of about 20° C. to about 40° C., at a gel-forming blockcopolymer concentration of about 4 wt. % or more, preferably about 4 wt.% to about 10 wt. %, based on total weight of the gel including solvent.Stiffness of the physical gels, represented by the storage modulus (G′),can vary from about 300 Pascals (Pa) to about 12,000 Pa by adjusting thegel-forming block copolymer structure and/or the concentration. Thesolvent can be water, an organic solvent, or a mixture thereof. Organicsolvents include volatile organic liquids (e.g., ethanol) and organicliquids having little or no volatility (e.g., mineral oils, plant oils)at standard temperatures and pressures.

Herein, “gels” means “hydrogels and/or organogels” unless otherwiseindicated. The gels are formed by noncovalent interactions of polymerchains of the gel-forming block copolymer in a given solvent. The gelnetwork is composed of physical crosslinks of these polymer chains. Thegels can serve as a matrix for delivering a variety of medically usefulmaterials, including one or more drugs, which can be physically loadedinto the gels.

Herein, a “drug” can be any substance recognized or defined by the U.S.Food, Drug, and Cosmetic Act that can be effectively formulated with thedisclosed gels and provide an effective therapeutic use. Drugs includesubstances used in the diagnosis, treatment, and/or prevention of adisease, and/or treatment of wounds. Drugs also include materials usedin topically applied cosmetics products and cosmetic surgery products.Drugs also include dietary products such as vitamins. Drugs also includeliving cells if used for a therapeutic treatment. The drugs arepreferably contained in the gel without being covalently bound to thegel-forming block copolymer. The drugs can be dissolved or dispersed inthe gel matrix.

A drug can be a naturally produced or synthetic compound, a naturallyproduced or synthetic polymer, or combinations of the foregoingmaterials. Non-limiting exemplary naturally produced compounds includepaclitaxel, artemisinin, alkaloids, terpenoids, phytosterols, naturalphenols, ciclosporin, lovastatin, morphine, quinine, tubocurarine,nicotine, muscarine, asperlicin, eleutherobin, discodermolide,bryostatins, dolostatins, cephalostatins, and vitamins. Exemplarysynthetic compounds include the antimicrobial drugs cephalosporins,tetracyclines, aminoglycosides, rifamycins, chloramphenicol, fluconazoleand doxycycline. Exemplary naturally produced polymers include genes,nucleotides, proteins, and peptides. Exemplary synthetic polymersinclude antimicrobial cationic polycarbonates and monoclonal antibodiesproduced artificially by a genetic engineering technique.

Herein, a vitamin is defined as any of a group of organic compounds thatare essential in small quantities for normal metabolism of a livingbody, and generally cannot be synthesized in the body. Exemplaryvitamins include vitamin A (retinol), vitamin B1 (thiamine), vitamin B2(riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid),vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid),vitamin B12 (cobalamines), beta-carotene, vitamin C (ascorbic acid),vitamin D compounds (which include vitamin D1 (calciferol), vitamin D2(ergocalciferol), and/or vitamin D3 (cholecalciferol)), vitamin Ecompounds (which include alpha-tocopherol, beta-tocopherol,gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol,gamma-tocotrienol, and/or delta-tocotrienol), and vitamin K1(phylloquinone).

Herein, a loaded gel is a medical composition comprising a solvent,about 2 wt. % to about 10 wt. % of a gel-forming block copolymer, andabout 0.0001 wt. % to about 10 wt. % of a drug, more specifically about0.0001 wt. % to about 2 wt. % of the drug, and even more specificallyabout 0.0001 wt. % to about 1 wt. % of the drug. For example, a loadedgel can comprise an anti-tumor drug suitable for treating a cancer. Asanother example, a loaded gel can comprise an antimicrobial cationicpolymer (a first drug). The antimicrobial cationic polymer can be ahomopolymer, random copolymer, block copolymer, star polymer, starpolymer having a crosslinked microgel core, dendritic polymer, or acombination of the foregoing polymer types. Preferably, theantimicrobial cationic polymer is one or more of the cationic polymersdescribed further below, which are cationic polycarbonates. Theantimicrobial cationic polycarbonate is preferably contained in the gelwithout being covalently bound to the gel-forming block copolymer. Theloaded gel can further comprise an antimicrobial compound (a seconddrug) such as, for example, fluconazole, which is suitable foreradicating biofilms. The loaded gels can include one or more drugs,which are associated by noncovalent interactions in the gel. The loadedgels provide a means for controlling the release rate of a drug andlocalizing the drug in the vicinity of the application site or injectionsite, thereby increasing the efficacy of the drug.

The loaded gel compositions can be delivered by various types ofinjection, including intradermal, subcutaneous, intramuscular,intravenous, intraosseous, and/or intraperitoneal injection. The loadedgels can be applied topically to a skin surface (e.g., for transdermaldelivery of biologically active substances), and/or to other bodysurfaces, including the eyes and body cavities. The loaded gels can alsobe applied to wounds.

The solvent for the loaded gel can be water and/or an organic solvent. Anon-limiting example of an organic solvent is KOLLIPHOR RH40 (registeredtrademark of BASF). KOLLIPHOR RH40, also known as PEG-40 castor oil, isa non-ionic polyethoxylated detergent.

The gel-forming block copolymers have a structure in accordance withformula (1):

wherein

d′ is a positive number having an average value of 100 to 600,

L′ and L″ are independent divalent linking groups, wherein L′ and/or L″comprises a divalent *—N(H)—* group,

each P^(a) is an independent monocarbonate or polycarbonate chaincomprising on average more than 0 and up to 10 vitamin-bearing subunits,wherein each of the vitamin-bearing subunits comprises a carbonatebackbone portion and a side chain linked to the carbonate backboneportion, the side chain comprising a covalently bound form of a vitamin,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons.

Preferably the vitamin-bearing subunits are non-charged. In anembodiment, each P^(a) consists essentially of 1 to about 10vitamin-bearing subunits.

The *—N(H)—* group of L′ and/or L″ is capable of donating an electronpair of the nitrogen and/or the hydrogen to form a hydrogen bond.Exemplary non-limiting nitrogen-bearing L′ and/or L″ groups include thefollowing:

In the above linking groups, atomic center labeled 1 is linked to thehydrophilic block, and atomic center labeled 2 of the linking group islinked to an end unit of one of the P^(a) groups. It should beunderstood that the nitrogen of linking group *—NH—*, when present, islinked to P^(a) and the hydrophilic block.

Herein, an atomic center shown linked to an asterisk (*-) indicates theatomic center is covalently linked to another unspecified atomic centerof a chemical structure represented by the asterisk. The bond to anasterisk is also referred to herein as a “starred bond”.

L′ or L″, but not both, can be *—O—* or a divalent alkyleneoxy grouphaving 2-10 carbons (e.g., ethyleneoxy (*—CH₂CH₂O—*), propoxy, butoxy,and the like). Preferably, each of L′ and L″ contains an *—NH—* groupcapable of donating an electron pair and/or hydrogen to form a hydrogenbond. In an embodiment, L′ is *—N(H)—* and L″ is

wherein carbon 1 is linked the terminal oxygen of hydrophilic block andnitrogen 2 is linked to one of the P^(a) groups. In another embodiment,L′ and L″ comprise an *—N(H)—* group, which is a portion of a carbamategroup.

The vitamin-bearing subunits of P^(a) can have a structure in accordancewith formula (2):

wherein

the carbonate backbone atoms are numbered 1 to 6,

L^(d) is a single bond or a divalent linking group comprising 1 to about15 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons,

t is a positive integer having a value of 0 to 2,

t′ is a positive integer having a value of 0 to 2, and

t and t′ cannot both be zero.

More specific vitamin-bearing subunits comprise a covalently bound formof a vitamin selected from the group consisting of vitamin D compounds,vitamin E compounds, and combinations thereof.

Still more specific vitamin-bearing subunits comprise a covalently boundform of a vitamin selected from the group consisting ofalpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol,alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol,delta-tocotrienol, and combinations thereof.

Even more specific vitamin-bearing subunits comprise a carbonatebackbone portion and a side chain linked to the backbone portion, theside chain comprising a covalently bound form of alpha-tocopherol (avitamin E compound):

a known antioxidant. The introduction of hydrophobic alpha-tocopherolmoieties into the A block has a significant influence on the thresholdconcentration for gelation and the rheological properties of thehydrogels. Moreover, alpha-tocopherol and poly(ethylene glycol) (PEG)are biocompatible and FDA-approved chemical compounds, which gives thehydrogel system added advantage in the evaluation of its use in apharmaceutical application.

The covalently bound form of the vitamin can be present as a singlestereoisomer or as a mixture of stereoisomers.

Z^(a) and Z^(b) are independent end groups. In an embodiment, each ofZ^(a) and Z^(b) is an acyl group (e.g., acetyl, benzoyl). In anotherembodiment, Z^(a) is hydrogen and Z^(b) is hydrogen.

A more specific vitamin-bearing subunit has a structure in accordancewith formula (3):

wherein

L^(d) is a single bond or a divalent linking group comprising 1 to about15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin, and

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons.

An even more specific vitamin-bearing subunit has a structure inaccordance with formula (4):

wherein

L^(e) is a single bond or a divalent linking group comprising 1 to about14 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin, and

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons.

In an embodiment, L^(e) comprises 1 to about 10 carbons.

Another more specific vitamin-bearing subunit has a structure inaccordance with formula (5):

wherein

L^(f) is a single bond or a divalent linking group comprising 1 to about14 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl, and

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons.

In an embodiment, L^(f) comprises 1 to about 10 carbons.

A more specific gel-forming block copolymer has a structure according toformula (1-A):

wherein

atoms numbered 1 to 6 of the carbonate subunits enclosed by parenthesesare backbone atoms of the block copolymer,

d′ is a positive number having an average value of 100 to 600,

each m′ is an independent positive number having an average value whichis greater than 0 and less than or equal to 10,

L′ and L″ are independent divalent linking groups, wherein L′ and/or L″comprises a divalent *—N(H)—* group,

each L^(d) is independently a single bond or a divalent linking groupcomprising 1 to 15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no carbonate subunit has t=0 and t′=0,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to 15 carbons.

In an embodiment, each R′ is hydrogen, each R″ is methyl or ethyl, eacht′ is 1, each t″ is 1, Z^(a) is hydrogen, and Z^(b) is hydrogen.

Another more specific gel-forming block copolymer has a structureaccording to formula (1-B):

wherein

atoms numbered 1 to 6 of the carbonate subunits enclosed by parenthesesare backbone atoms of the block copolymer,

d′ is a positive number having an average value of 100 to 600,

each m′ is an independent positive number having an average value whichis greater than 0 and less than or equal to 10,

L′ and L″ are independent divalent linking groups, wherein L′ and/or L″comprises a divalent *—N(H)—* group,

each L^(e) is independently a single bond or a divalent linking groupcomprising 1 to about 15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons.

In an embodiment, each R″ is methyl or ethyl, Z^(a) is hydrogen, andZ^(b) is hydrogen.

Another more specific gel-forming block copolymer has a structureaccording to formula (1-C):

wherein

atoms numbered 1 to 6 of the carbonate subunits enclosed by parenthesesare backbone atoms of the block copolymer,

d′ is a positive number having an average value of 100 to 600,

each m′ is an independent positive number having an average value whichis greater than 0 and less than or equal to 10,

each L^(d) is independently a single bond or a divalent linking groupcomprising 1 to about 15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons.

In an embodiment, each R″ is methyl.

Another more specific gel-forming block copolymer has a structureaccording to formula (1-D):

wherein

atoms numbered 1 to 6 of the carbonate subunits enclosed by parenthesesare backbone atoms of the block copolymer,

d′ is a positive number having an average value of 100 to 600,

each m′ is an independent positive number having an average value whichis greater than 0 and less than or equal to 10,

each L^(e) is independently a single bond or a divalent linking groupcomprising 1 to about 15 carbons,

each V′ is an independent moiety comprising a covalently bound form of avitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen and alkyl groups comprising 1 to 6 carbons,

Z^(a) is a first end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons, and

Z^(b) is a second end group selected from the group consisting ofhydrogen and groups comprising 1 to about 15 carbons.

In an embodiment, each R″ is methyl. In another embodiment, d′ has anaverage value in a range of 200 to 500. In another embodiment, d′ has anaverage value in a range of 200 to 500.

A preferred method of preparing the gel-forming block copolymersutilizes an organocatalyzed ring opening polymerization of a cycliccarbonate monomer that comprises a covalently bound form of a vitamin,referred to as a vitamin-bearing monomer. The ring openingpolymerization (ROP) can be initiated by a ROP polymeric initiatorcomprising a poly(ethylene oxide) chain and at least one end groupcomprising a nitrogen, which after the ROP is present in an *—N(H)—*group capable of forming a hydrogen bond by way of an electron pair ofthe nitrogen and/or by way of the hydrogen covalently linked to thenitrogen. The ROP polymeric initiator can have nucleophilic alcohol,amine, and/or thiol group for initiating the ROP. The ROP polymericinitiator can have a number average molecular weight (Mn) of about 5000to about 25,000, more particularly 10,000 to about 20,000. Thevitamin-bearing monomers can also be used for the preparation of theantimicrobial cationic copolymers described further below.

The vitamin bearing monomers can have a structure according to formula(6):

wherein

the ring atoms are shown numbered 1 to 6,

L^(d) is a single bond or a divalent linking group comprising 1 to about15 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons,

t is a positive integer having a value of 0 to 2,

t′ is a positive integer having a value of 0 to 2, and

t and t′ cannot both be zero.

Ring opening polymerization of vitamin-bearing monomers of formula (6)produces an initial polycarbonate having a vitamin-bearing subunit offormula (2) described further above.

More specific vitamin bearing monomers can have a structure according toformula (7):

wherein

the ring atoms are shown numbered 1 to 6,

L^(d) is a single bond or a divalent linking group comprising 1 to about15 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin, and

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons.

Ring opening polymerization of vitamin-bearing monomers of formula (7)produces an initial polycarbonate having a vitamin-bearing subunit offormula (3) described further above. In an embodiment, R″ at carbon 5 isselected from the group consisting of hydrogen, methyl, and ethyl.

Other more specific vitamin-bearing monomers can have a structureaccording to formula (8):

wherein

ring atom 5 is labeled,

L^(e) is a single bond or a divalent linking group comprising 1 to about14 carbons,

V′ is a moiety comprising a covalently bound form of a vitamin, and

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons.

Ring opening polymerization of vitamin-bearing monomers of formula (8)produces an initial polycarbonate having a vitamin-bearing subunit offormula (4) described further above.

The cyclic carbonate monomers used in the ROP can be stereospecific ornon-stereospecific.

An exemplary compound of formula (8) is MTC-VitE:

having a pendent alpha-tocopheryl group. MTC-VitE undergoes a ringopening polymerization forming a carbonate subunit having the structure:

Another compound of formula (8) is MTC-VitD2:

which has a pendent ergocalciferyl group. MTC-VitD2 undergoes a ringopening polymerization forming a carbonate subunit having the structure:

Other vitamin-bearing monomers can have a structure according to formula(9):

wherein

the ring atoms are shown numbered 1 to 6,

L^(f) is a single bond or a divalent linking group comprising 1 to about14 carbons,

V′ is a covalently bound form of a vitamin, and

R″ is a monovalent radical selected from the group consisting ofhydrogen and alkyl groups comprising 1 to 6 carbons.

Ring opening polymerization of vitamin-bearing monomers of formula (9)produces an initial polycarbonate having a vitamin-bearing subunit offormula (5) described further above.

Several preferred types of antimicrobial cationic polymers are describedin the following section.

Antimicrobial cationic polymers having one polymer chain (one-armed)

A first antimicrobial cationic polymer has a structure in accordancewith formula (10):Z′—P′—Z″  (10),wherein

Z′ is a monovalent C₁-C₁₅ first end group, wherein Z′ is linked to abackbone carbonyl group of P′,

Z″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

P′ is a polymer chain consisting essentially of cationic carbonatesubunits, wherein i) P′ has a degree of polymerization (DP) of about 5to about 45, ii) each of the cationic carbonate subunits comprises abackbone portion of the polymer chain and a C₆-C₂₅ cationic side chainlinked to the backbone portion, and iii) the cationic side chaincomprises a positive-charged heteroatom Q′ of a quaternary ammoniumgroup and/or quaternary phosphonium group,

about 25% to about 100% of the cationic carbonate subunits, designatedfirst cationic carbonate subunits, have a cationic side chain comprising13 to about 25 carbons, and

about 0% to about 75% of the cationic carbonate subunits, designatedsecond cationic carbonate subunits, have a cationic side chaincomprising 6 to 12 carbons.

Z′ can be any suitable end group comprising 1 to 15 carbons. Z′comprises an oxygen, nitrogen or sulfur heteroatom that is linked to abackbone carbonyl of P′ in the form of a carbonate, carbamate orthiocarbonate group, respectively. Z′ can be a residue of an initiatorused in a ring opening polymerization to form the cationic polymer. Inan embodiment, Z′ is a covalently bound form of C₁-C₁₅ compound. Inanother embodiment, Z′ is a C₁-C₁₅ alkoxy or aryloxy group.

Z″ is preferably linked to a backbone oxygen of P′. When Z″ is hydrogen,the cationic polymer has a terminal hydroxy group. When Z″ is nothydrogen, Z″ can be any suitable end group comprising 1 to 15 carbons.In an embodiment, Z″ is a covalently bound form of C₁-C₁₅ compound. Inanother embodiment, Z″ is a C₁-C₁₅ acyl group.

The first cationic carbonate subunits preferably comprise a cationicside chain having 13 to about 20 carbons, even more preferably 15 toabout 20 carbons.

In an embodiment, P′ consists essentially of 25 mol % to about 75 mol %of the first cationic carbonate subunits and about 75 mol % to about 25mol % of the second cationic carbonate subunits. In another embodiment,P′ consists essentially of 25 mol % to about 50 mol % of the firstcationic carbonate subunits and about 75 mol % to about 25 mol % of thesecond cationic carbonate subunits.

The cationic carbonate subunits can have a structure according toformula (11):

wherein

atoms labeled 1-6 are atoms of the polymer backbone,

L^(a)-Q′(R^(a))_(u′) is a C₆-C₂₅ cationic side chain comprising aquaternary ammonium group and/or quaternary phosphonium group, whereinL^(a) is a divalent linking group comprising at least 3 carbons, Q′ is atetravalent positive-charged nitrogen or phosphorus, u′ has a value of 1to 3, each R^(a) is an independent radical having a valency of 1 to 3,and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons,

t is a positive integer having a value of 0 to 2,

t′ is a positive integer having a value of 0 to 2,

t and t′ cannot both be zero, and

X′ is a negative-charged ion.

In an embodiment, t and t′ are both 1, each R′ is hydrogen, and R″ ismethyl or ethyl.

In a cationic polymer of formula (10) whose cationic carbonate subunitsare of formula (11), each of the first cationic carbonate subunits has acationic side chain L^(a)-Q′(R^(a))_(u′) comprising 13 to about 25carbons, and each of the second cationic carbonate subunits has acationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 12 carbons.

More specific cationic subunits have a structure in accordance withformula (12):

wherein

L^(b)-Q′(R^(a))_(u′) is a C₅-C₂₄ cationic moiety comprising a quaternaryammonium group and/or quaternary phosphonium group, wherein L^(b) is adivalent linking group comprising at least 2 carbons, Q′ is atetravalent positive-charged nitrogen or phosphorus, u′ has a value of 1to 3, each R^(a) is an independent radical having a valency of 1 to 3,and each R^(a) comprises at least 1 carbon,

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons, and

X′ is a negative-charged ion.

In this instance, the cationic side chain group isC(═O)O-L^(b)-Q′(R^(a))_(u′) and C(═O)O-L^(b) corresponds to divalentlinking group L^(a) of formula (11). The cationic side chain is linkedto backbone carbon labeled 5.

In a cationic polymer of formula (10) whose cationic carbonate subunitsare of formula (12), each of the first cationic carbonate subunits has acationic side chain C(═O)O-L^(b)-Q′(R^(a))_(u′) comprising 13 to about25 carbons, and each of the second cationic carbonate subunits has acationic side chain C(═O)O-L^(b)-Q′(R^(a))_(u′) comprising 6 to 12carbons.

Another more specific cationic subunit has a structure in accordancewith formula (13):

wherein

L^(c)-Q′(R^(a))_(u′) is a C₅-C₂₄ cationic moiety comprising a quaternaryammonium group and/or quaternary phosphonium group, wherein L is adivalent linking group comprising at least 2 carbons, Q′ is atetravalent positive-charged nitrogen or phosphorus, u′ has a value of 1to 3, and each R^(a) is an independent radical having a valency of 1 to3, wherein each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons, and

X′ is a negative-charged ion.

In this instance the cationic side chain isN(H)C(═O)O-L^(c)-Q′(R^(a))_(u′) and N(H)C(═O)O-L^(c) corresponds todivalent linking group L^(a) of formula (11). The cationic side chain islinked to backbone carbon labeled 5. Serinol and/or threoninol provideuseful starting materials for the formation of subunits of formula (13).

In a cationic polymer of formula (10) whose cationic carbonate subunitsare of formula (13), each of the first cationic carbonate subunits has acationic side chain N(H)C(═O)O-L^(c)-Q′(R^(a))_(u′) comprising 13 toabout 25 carbons, and each of the second cationic carbonate subunits hasa cationic side chain N(H)C(═O)O-L^(c)-Q′(R^(a))_(u′) comprising 6 to 12carbons.

Using the cationic subunit of formula (11), the cationic polymers offormula (10) can have a structure in accordance with formula (14):

wherein:

n′ represents the number of cationic carbonate subunits, wherein n′ hasa value of about 5 to about 45,

Z′ is a monovalent C₁-C₁₅ first end group,

Z″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2, nocationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 13 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 12 carbons.

As shown in formula (14), the polymer chain comprises a backbone portioncomprising an oxycarbonyl group at a first end of the chain (referred toas the “carbonyl end”), and a backbone oxygen at a second end of thechain (referred to as the “oxy end”). The backbone atoms of the cationiccarbonate subunit are shown numbered 1 to 6.

In formula (14), L^(a) and Q′(R^(a))_(u′) of the first cationiccarbonate subunits can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 13 to about 25 carbons.Preferably, the L^(a) group of the first cationic carbonate subunitscomprises 5 to about 12 carbons, or more preferably 8 to about 12carbons. Preferably, Q′(R^(a))_(u′) of the first cationic carbonatesubunits comprise 3 to about 18 carbons, more preferably 4 to about 18carbons.

Likewise, L^(a) and Q′(R^(a))_(u′) of the second cationic carbonatesubunits of formula (14) can each have at least 3 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 6 to 12 carbons.

In an embodiment, Z″ is hydrogen. In another embodiment, the firstcationic carbonate subunits have a cationic side chainL^(a)-Q′(R^(a))_(u′) comprising 15 to about 20 carbons.

As more specific non-limiting examples, Z′ can be benzyloxy and/or4-methylbenzyloxy, and Z″ can be hydrogen and/or acetyl.

The end groups Z′ and/or Z″, and end groups described below, can enhanceantimicrobial efficacy and or stabilize the cationic polymer frompotential unwanted side reactions (e.g., chain scission) caused by, forexample, an unblocked nucleophilic hydroxy end group. Bulkier end groupscan also provide hydrophobicity allowing control of the amphiphilicproperties of the cationic polymers.

The antimicrobial cationic polymers can have a structure in accordancewith formula (15):

wherein

n′ represents the number of cationic carbonate subunits, and n′ has avalue of about 5 to about 45,

Y′ is a monovalent first end group comprising a covalently bound form ofa biologically active compound selected from the group consisting ofsteroids, non-steroid hormones, vitamins, and drugs,

Y″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (15) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (15) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

The biologically active compound can be stereospecific ornon-stereospecific. In an embodiment, Y′ comprises a covalently boundform of a steroid (e.g., cholesterol), designated S′. The steroid groupcan enhance biocompatibility of the cationic polymer.

In another embodiment, Y′ comprises a covalently bound form of avitamin, (e.g., alpha-tocopherol (a vitamin E compound) and/orergocalciferol (vitamin D2)).

Y′ can have a structure S′-L′-* wherein S′ is a steroid group and L′ isa single bond or any suitable divalent linking group comprising 1 toabout 10 carbons. In this instance, L′ links S′ to the carbonyl end ofthe polycarbonate backbone.

The antimicrobial cationic polymers can have a structure in accordancewith formula (16):

wherein

n′ represents the number of cationic carbonate subunits, and n′ has avalue of about 5 to about 45,

W′ is a monovalent C₁-C₁₅ first end group,

W″ is a monovalent second end group comprising a covalently bound formof a biologically active compound selected from the group consisting ofsteroids, non-steroid hormones, vitamins, and drugs,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2, nocationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (16) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (16) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

W″ can comprise a stereospecific or non-stereospecific form of thebiologically active compound. In an embodiment, W″ comprises acovalently bound form of cholesterol, alpha-tocopherol (a vitamin Ecompound), ergocalciferol (vitamin D2), or combinations thereof.

W″ can have the general structure S′-L″-* wherein S′ is a steroid groupand L″ is a single bond or any suitable divalent linking groupcomprising 1 to about 10 carbons. In this instance, L″ links S′ to theoxy end of the polycarbonate backbone.

The antimicrobial cationic polymer can be a random copolymer having astructure in accordance with formula (17):Z′—P″—Z″  (17),wherein

Z′ is a monovalent C₁-C₁₅ first end group,

Z″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

P″ is a polymer chain consisting essentially of I) about 85 mol % to99.9 mol % of cationic carbonate subunits, and II) 0.1 mol % to about 15mol % of carbonate subunits comprising a covalently bound form of asteroid and/or a vitamin compound, wherein i) P″ has a degree ofpolymerization (DP) of about 5 to about 45, ii) each of cationiccarbonate subunits comprises a polymer backbone portion and a cationicside chain portion linked to the polymer backbone portion, and iii) eachcationic side chain portion comprises a positively charged heteroatom ofa quaternary ammonium group and/or a quaternary phosphonium group,

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

More specific antimicrobial cationic polymers of formula (17) can have astructure in accordance with formula (18):

wherein

n′ represents the number of cationic carbonate subunits, wherein n′ hasa value greater than 0,

m′ represents the number of carbonate subunits, wherein m′ has a valuegreater than 0,

n′+m′ has a value of about 5 to about 45,

a ratio of m′:n′ is about 15:85 to about 0.1:99.9,

Z′ is a monovalent C₁-C₁₅ first end group,

Z″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

each L^(d) is an independent divalent linking group selected from thegroup consisting of single bond and monovalent radicals comprising 1 toabout 10 carbons,

each H′ is an independent monovalent radical comprising a covalentlybound form of a steroid and/or a vitamin compound,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

The square brackets of formula (18) enclose a polymer chain. Thevertical stacking of subunits within the square brackets indicates arandom distribution of subunits within the polymer chain.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (18) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (18) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

H′ can comprise a covalently bound form of a vitamin E compound, vitaminD compound, or combinations thereof. Preferably, the vitamin compound isalpha-tocopherol (a vitamin E compound), ergocalciferol (vitamin D2), ora combination thereof.

The discussion that follows applies generally to all of theabove-described cationic polymers.

Exemplary non-limiting divalent L^(a) groups include:

and combinations thereof. In these examples, the starred bonds of thecarbonyl and carbamate nitrogen are linked to the polycarbonate backbone(e.g., the backbone carbon labeled 5 in the above cationic carbonatesubunits), and the starred bonds of the methylene groups are linked toQ′.

Together, L^(a) and Q′(R^(a))_(u′) form a quaternary ammonium group or aquaternary phosphonium group, meaning the positive-charged heteroatom Q′is bonded to a carbon of La and up to three independent R^(a) groups.

Each R^(a) comprises at least one carbon. Each R^(a) can be a monovalenthydrocarbon substituent (e.g., methyl, ethyl, etc.), in which case u′ is3.

An R^(a) can form a ring with Q′, in which case the R^(a) of the ringhas a valency of 2. For example, Q′(R^(a))_(u′) can be:

wherein the starred bond is linked to L^(a), Q′ is nitrogen, and u′ is2. In this example, a first R^(a) is a divalent butylene group(*—(CH₂)₄—*), and a second R^(a) is methyl.

R^(a) can form a multi-cyclic moiety with Q′. For example Q′(R^(a))_(u′)can be:

wherein the starred bond is linked to L^(a), Q′ is nitrogen, u′ is 1,and R^(a) is the fragment

having a valency of 3.

The R^(a) groups can also independently comprise oxygen, nitrogen,sulfur, and/or another heteroatom. In an embodiment, each R^(a) is anindependent monovalent branched or unbranched hydrocarbon substituent.

Exemplary non-limiting R^(a) groups include methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,and benzyl.

Exemplary non-limiting Q′(R^(a))_(u′) groups include:

and combinations thereof.

In the foregoing examples, it should be understood that thepositive-charged nitrogen and phosphorus are tetravalent, and thestarred bond is linked to a carbon of L^(a). The Q′ groups can bepresent in the cationic polymer singularly or in combination.

Exemplary negative-charged ions X′ include halides (e.g., chloride,bromide, and iodide), carboxylates (e.g., acetate and benzoate), and/orsulfonates (e.g., tosylate). The X′ ions can be present singularly or incombination.

Exemplary non-limiting cationic carbonate subunits include thefollowing:

and combinations thereof, wherein X is a negative-charged ion associatedionically with the cation.

In general, antimicrobial activity of the cationic polymers is favoredby spacing the positive-charged heteroatom Q′ from the polycarbonatebackbone in 25 mol % to 100 mol % of the cationic carbonate subunits(first cationic carbonate subunits) by the shortest path having 6 ormore contiguously linked atomic centers from the polymer backbone. Theshortest path is defined as the lowest number of contiguously linkedatomic centers joining Q′ to the polymer backbone. The contiguouslylinked atomic centers should be understood to be between thepolycarbonate backbone and Q′. For example, if L^(a)-Q′ is:

then the shortest path from the polymer backbone to Q′ has 5contiguously linked atomic centers, as numbered. The shortest path doesnot include the carbonyl oxygen. As another example, if L^(a)-Q′ is

then the shortest path from the polymer backbone to Q′ has 6contiguously linked atomic centers, as numbered. The shortest path doesnot include the amide hydrogen and the carbonyl oxygen. As anotherexample, if L^(a)-Q′ is

then the shortest path from the polymer backbone to Q′ has 8contiguously linked atomic centers, as numbered. The shortest path doesnot include two carbons of the aromatic ring and the carbonyl oxygen. Asanother example, if L^(a)-Q′ is

then the shortest path from the polymer backbone to Q′ has 7contiguously linked atomic centers, as numbered. The shortest path doesnot include three carbons of the aromatic ring and the carbonyl oxygen.Finally, as another example, if L^(a)-Q′ is

then the shortest path from the polymer backbone to Q′ has 4contiguously linked atomic centers, as numbered. The shortest path doesnot include the aromatic ring and the carbonyl oxygen.

Preferably, Q′ of the first carbonate subunits is spaced from thepolymer backbone by the shortest path having 6 to about 15 contiguouslylinked atomic centers, and more preferably 8 to about 15 contiguouslylinked atomic centers.

The steroid group S′ can originate from a naturally occurring humansteroid, non-human steroid, and/or a synthetic steroid compound. Herein,a steroid group comprises a tetracyclic ring structure:

wherein the 17 carbons of the ring system are numbered as shown. Thesteroid group can comprise one or more additional substituents attachedto one or more of the numbered ring positions. Each ring of thetetracyclic ring structure can independently comprise one or more doublebonds.

Exemplary steroid groups include cholesteryl, from cholesterol, shownbelow without stereochemistry:

Non-limiting stereospecific structures of cholesteryl include

where the R,S stereoconfiguration of each stereospecific asymmetriccenter is labeled.

Additional non-limiting steroid groups include

from testosterone,

from equilin,

from epiandrosterone,

from dihydrotestosterone,

from nandrolone,

from dihydroprogesterone,

from pregnenolone,

from equilenin,

from dehydroepiandrosterone,

from corticosterone acetate,

from deoxycorticosterone, and

from estrone.

The starred bonds represent attachment points. For example, the starredbond of each of the above steroid groups can be linked to a terminalcarbonyl group of the polycarbonate backbone by way of a divalentlinking group L′. Alternatively, the starred bond of the steroid groupcan be directly linked to a terminal carbonyl group of the polycarbonatebackbone (i.e., L′ can be a single bond).

Those of skill in the art will recognize that each asymmetric center ofthe steroid groups can be present as the R stereoisomer, S stereoisomer,or as a mixture of R and S stereoisomers. Additional steroid groups S′include the various stereoisomers of the above structures. The cationicpolymer can comprise a steroid group as a single stereoisomer or as amixture of stereoisomers.

In an embodiment, S′ is cholesteryl group, wherein the cholesteryl groupis a mixture of isomers

indicated by the structure

More specific steroid-containing cationic polymers have a structure inaccordance with formula (19):

wherein

n′ represents the number of cationic carbonate subunits, and has a valueof about 5 to about 45,

S′-L′ is a first end group, wherein L′ is a single bond or a divalentlinking group comprising 1 to about 10 carbons, and S′ comprises acovalently bound form of a steroid,

Y″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

La and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (19) can individually have 3 to about 22 carbons, with theproviso that La-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons. Inan embodiment, each of the first cationic carbonate subunits of formula(19) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 13 toabout 25 carbons, and each of the second cationic carbonate subunits hasa cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 12 carbons.

In formula (19), when L′ is a single bond, S′ is linked directly to theterminal carbonyl group of the polycarbonate backbone. In an embodiment,L′ is a divalent linking group comprising an alkylene oxide selectedfrom the group consisting of ethylene oxide (*—CH₂CH₂O—*), propyleneoxide *—CH₂CH₂CH₂O—*, and/or tri(ethylene oxide)(*—CH₂CH₂OCH₂CH₂OCH₂CH₂O—*), wherein the starred bond of the oxygen islinked to the terminal carbonyl group of the polycarbonate backbone andthe starred bond of the carbon is linked to S′.

The steroid-containing cationic polymers can comprise one or acombination of the cationic carbonate subunits described further above.

The steroid-containing cationic polymers can have a structure inaccordance with formula (20):

wherein

n′ represents the number of cationic carbonate subunits, and has a valueof about 5 to about 45,

Y′ is a monovalent C₁-C₁₅ first end group,

S′-L″ is a second end group, wherein L″ is a single bond or a divalentlinking group comprising 1 to about 10 carbons and S′ comprises acovalently bound form of a steroid,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

The group S′-L″ is linked to the oxy end of the polycarbonate backbone,and Y′ is linked to the carbonyl end of the polycarbonate backbone.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (20) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (20) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

Cationic polymers having two cationic polymer chains (two-armed cationicpolymers)

The antimicrobial cationic polymers can have a structure in accordancewith formula (21):Z^(c)—P^(b)—C′—P^(b)—Z^(c)  (21),wherein

C′ is a C₂-C₁₅ divalent linking group joining polymer chains P^(b),wherein C′ comprises i) a first heteroatom linked to a first polymerchain P^(b), wherein the first heteroatom is selected from the groupconsisting of nitrogen, oxygen, and sulfur, and ii) a second heteroatomlinked to a second polymer chain P^(b), wherein the second heteroatom isselected from the group consisting of nitrogen, oxygen, and sulfur,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each polymer chain P^(b) consists essentially of cationic carbonatesubunits, wherein i) the cationic polymer comprises a total of 5 toabout 45 cationic carbonate subunits, ii) each of the cationic carbonatesubunits comprises a backbone portion of the polymer chain and acationic side chain linked to the backbone portion, and iii) thecationic side chain comprises a positive-charged heteroatom Q′ of aquaternary ammonium group and/or quaternary phosphonium group,

about 25% to 100% of all the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (21) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (21) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

In an embodiment, each Z^(c) is hydrogen. In another embodiment, thepositive-charged heteroatom Q′ of the first cationic carbonate subunitsis spaced from the backbone portion by the shortest path having 6 toabout 15 contiguously linked atomic centers between Q′ and the backboneportion.

More specific cationic polymers of formula (21) have a structureaccording to formula (22):

wherein

n′ represents the total number of cationic carbonate subunits of thecationic polymer, and has a value of about 5 to about 45,

C′ is a C₂-C₁₅ divalent linking group joining polymer chains P^(b),wherein C′ comprises i) a first heteroatom linked to a first polymerchain P^(b), wherein the first heteroatom is selected from the groupconsisting of nitrogen, oxygen, and sulfur, and ii) a second heteroatomlinked to a second polymer chain P^(b), wherein the second heteroatom isselected from the group consisting of nitrogen, oxygen, and sulfur,

the polymer chains P^(b) consist essentially of the cationic carbonatesubunits,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2, nocationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (22) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (22) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

C′ can be a residue of a non-polymeric di-nucleophilic initiator used toprepare the cationic polymer by ring opening polymerization.

In another antimicrobial polymer, the fragment linking the two cationicpolymer chains comprises a covalently bound form of a biologicallyactive compound selected from the group consisting of steroids,non-steroid hormones, vitamins, and drugs. These antimicrobial cationicpolymers have a structure in accordance with formula (23):Z^(c)—P^(b)—C″—P^(b)—Z^(c)  (23),wherein

C″ is a divalent linking group joining polymer chains P^(b), wherein C″comprises i) a first heteroatom linked to a first polymer chain P^(b),wherein the first heteroatom is selected from the group consisting ofnitrogen, oxygen, and sulfur, ii) a second heteroatom linked to a secondpolymer chain P^(b), wherein the second heteroatom is selected from thegroup consisting of nitrogen, oxygen, and sulfur, and iii) a covalentlybound form of a compound selected from the group consisting of steroids,non-steroid hormones, vitamins, and drugs,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each polymer chain P^(b) consists essentially of cationic carbonatesubunits, wherein i) the cationic polymer comprises a total of 5 toabout 45 cationic carbonate subunits, ii) each of the cationic carbonatesubunits comprises a backbone portion of the polymer chain and a C₆-C₂₅cationic side chain linked to the backbone portion, and iii) thecationic side chain comprises a positive-charged heteroatom Q′ of aquaternary ammonium group and/or quaternary phosphonium group,

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain group comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain group comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (23) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (23) has a cationic side chain comprising 13 to about 25carbons, and each of the second cationic carbonate subunits has acationic side chain comprising 6 to 12 carbons.

The positive-charged heteroatom Q′ of the first cationic carbonatesubunits can be spaced from the backbone portion by the shortest pathhaving 6 to about 18 contiguously linked atomic centers between Q′ andthe backbone portion.

In an embodiment, C″ comprises a covalently bound form of cholesterol.In another embodiment, C″ comprises a covalently bound form of a vitaminselected from the group consisting of alpha-tocopherol, ergocalciferol,and combinations thereof.

More specific cationic polymers of formula (23) have a structureaccording to formula (24):

wherein

n′ represents the total number of cationic carbonate subunits of thecationic polymer, and has a value of about 5 to about 45,

C″ is a divalent linking group joining polymer chains P^(b), wherein C″comprises i) a first heteroatom linked to a first polymer chain P^(b),wherein the first heteroatom is selected from the group consisting ofnitrogen, oxygen, and sulfur, ii) a second heteroatom linked to a secondpolymer chain P^(b), wherein the second heteroatom is selected from thegroup consisting of nitrogen, oxygen, and sulfur, and iii) a covalentlybound form of a compound selected from the group consisting of steroids,non-steroid hormones, vitamins, and drugs,

each of the polymer chains P^(b) consists essentially of cationiccarbonate subunits,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2, nocationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (24) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (24) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

The antimicrobial cationic polymers can have a structure in accordancewith formula (25):Z^(c)—P^(c)—C′—P^(c)—Z^(c)  (25),wherein

C′ is a C₂-C₁₅ divalent linking group joining polymer chains P^(c),wherein C′ comprises i) a first heteroatom linked to a first polymerchain P^(c), wherein the first heteroatom is selected from the groupconsisting of nitrogen, oxygen, and sulfur, and ii) a second heteroatomlinked to a second polymer chain P^(c), wherein the second heteroatom isselected from the group consisting of nitrogen, oxygen, and sulfur,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each P^(c) is a polymer chain consisting essentially of I) about 85 mol% to 99.9 mol % of cationic carbonate subunits, and II) 0.1 mol % toabout 15 mol % of carbonate subunits comprising a covalently bound formof a steroid and/or a vitamin compound, wherein i) the cationic polymerhas a total number of subunits of about 5 to about 45, ii) each of thecationic carbonate subunits comprises a polymer backbone portion and aC₆-C₂₅ cationic side chain portion linked to the polymer backboneportion, and iii) each cationic side chain portion comprises apositive-charged heteroatom Q′ of a quaternary ammonium group and/orquaternary phosphonium group,

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain group comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain group comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (25) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (25) has a cationic side chain comprising 13 to about 25carbons, and each of the second cationic carbonate subunits has acationic side chain comprising 6 to 12 carbons.

The cationic polymers of formula (25) can have a structure according toformula (26):

wherein

n′ represents the total number of cationic carbonate subunits, whereinn′ has a value greater than 0,

m′ represents the total number of carbonate subunits, wherein m′ has avalue greater than 0,

n′+m′ has a value of about 5 to about 45, and

the ratio m′:n′ is about 15:85 to about 0.1:99.9,

C′ is a C₂-C₁₅ non-polymeric divalent linking group joining polymerchains P^(c), wherein C′ comprises i) a first heteroatom linked to afirst polymer chain P^(c), wherein the first heteroatom is selected fromthe group consisting of nitrogen, oxygen, and sulfur, and ii) a secondheteroatom linked to a second polymer chain P^(c), wherein the secondheteroatom is selected from the group consisting of nitrogen, oxygen,and sulfur,

each Z^(c) is an independent monovalent end group selected from thegroup consisting of hydrogen and C₁-C₁₅ moieties,

each L^(d) is an independent divalent linking group selected from thegroup consisting of single bond and monovalent radicals comprising 1 toabout 10 carbons,

each H′ is an independent monovalent radical comprising a covalentlybound form of a steroid and/or a vitamin compound,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (26) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (26) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

H′ can comprise a covalently bound form of a vitamin E compound, vitaminD compound, or combinations thereof. In an embodiment, H′ comprises acovalently bound form of a vitamin compound selected from the groupconsisting of alpha-tocopherol (a vitamin E compound), ergocalciferol(vitamin D2), and combinations thereof.

The antimicrobial cationic polymers can have a structure in accordancewith formula (27):Y^(c)—P^(b)—C′—P^(b)—Y^(d)  (27),wherein

C′ is a C₂-C₁₅ divalent linking group joining polymer chains P^(b),wherein C′ comprises i) a first heteroatom linked to a first polymerchain P^(b), wherein the first heteroatom is selected from the groupconsisting of nitrogen, oxygen, and sulfur, and ii) a second heteroatomlinked to a second polymer chain P^(b), wherein the second heteroatom isselected from the group consisting of nitrogen, oxygen, and sulfur,

Y^(c) is a monovalent first end group selected from the group consistingof hydrogen, groups comprising a covalently bound form of a steroid, andgroups comprising a covalently bound form of a vitamin,

Y^(d) is a monovalent second end group selected from the groupconsisting of hydrogen, groups comprising a covalently bound form of asteroid, and groups comprising a covalently bound form of a vitamin,

each polymer chain P^(b) consists essentially of cationic carbonatesubunits, wherein i) the cationic polymer comprises a total of 5 toabout 45 cationic carbonate subunits, ii) each of the cationic carbonatesubunits comprises a backbone portion of the polymer chain and acationic side chain linked to the backbone portion, and iii) thecationic side chain comprises a positive-charged heteroatom Q′ of aquaternary ammonium group and/or quaternary phosphonium group,

about 25% to 100% of all the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (27) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (27) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

Y^(c) and/or Y^(d) can comprise a covalently bound form of a steroidand/or a vitamin.

More specific cationic polymers of formula (27) have a structureaccording to formula (28):

wherein

n′ represents the total number of cationic carbonate subunits of thecationic polymer, and has a value of about 5 to about 45,

C′ is a C₂-C₁₅ divalent linking group joining polymer chains P^(b),wherein C′ comprises i) a first heteroatom linked to a first polymerchain P^(b), wherein the first heteroatom is selected from the groupconsisting of nitrogen, oxygen, and sulfur, and ii) a second heteroatomlinked to a second polymer chain P^(b), wherein the second heteroatom isselected from the group consisting of nitrogen, oxygen, and sulfur,

the polymer chains P^(b) consist essentially of the cationic carbonatesubunits,

Y^(c) is an independent monovalent end group selected from the groupconsisting of hydrogen, groups comprising a covalently bound form of asteroid, and groups comprising a covalently bound form of a vitamin,

Y^(d) is an independent monovalent end group selected from the groupconsisting of hydrogen, groups comprising a covalently bound form of asteroid, and groups comprising a covalently bound form of a vitamin,

each L^(a)-Q′(R^(a))_(u′) is an independent C₆-C₂₅ cationic side chaincomprising a quaternary ammonium group and/or quaternary phosphoniumgroup, wherein L^(a) is a divalent linking group comprising at least 3carbons, Q′ is a tetravalent positive-charged nitrogen or phosphorus, u′has a value of 1 to 3, each R^(a) is an independent radical having avalency of 1 to 3, and each R^(a) comprises at least 1 carbon,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2,

no cationic carbonate subunit has t=0 and t′=0, and

each X′ is an independent negative-charged ion;

and wherein

about 25% to 100% of the cationic carbonate subunits of the cationicpolymer, designated first cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 10 to about 25 carbons, and

0% to about 75% of the cationic carbonate subunits of the cationicpolymer, designated second cationic carbonate subunits, have a cationicside chain L^(a)-Q′(R^(a))_(u′) comprising 6 to 9 carbons.

L^(a) and Q′(R^(a))_(u′) of the first cationic carbonate subunits offormula (28) can individually have 3 to about 22 carbons, with theproviso that L^(a)-Q′(R^(a))_(u′) has a total of 10 to about 25 carbons.In an embodiment, each of the first cationic carbonate subunits offormula (28) has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising13 to about 25 carbons, and each of the second cationic carbonatesubunits has a cationic side chain L^(a)-Q′(R^(a))_(u′) comprising 6 to12 carbons.

Cation-Forming Cyclic Carbonate Monomers

A preferred method of preparing the disclosed cationic polymers utilizesa cyclic carbonate monomer capable of forming a cationic moiety beforeor after the polymerization. These are referred to as cation-formingmonomers, which have the formula (29):

wherein

the ring atoms are shown numbered 1 to 6,

L^(a) is a divalent linking group comprising at least 3 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to La, wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises 1 or more carbons, and together Q′(R^(a))_(u′) and L^(a)comprise 6 to about 25 carbons,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons,

t is a positive integer having a value of 0 to 2,

t′ is a positive integer having a value of 0 to 2, and

t and t′ cannot both be zero.

The cation-forming monomers of formula (29) have a ring substituentL^(a)-E′. This ring substituent L^(a)-E′ becomes a side chain of theinitial polymer formed by the ring opening polymerization of thecation-forming monomer. E′ can be an electrophilic and/or nucleophilicgroup so long as the side chain L^(a)-E′ is capable of reacting toproduce a C₆-C₂₅ cationic side chain L^(a)-Q′(R^(a))_(u′) of thecationic polymer. Preferably, E′ is a leaving group capable of reactingwith a tertiary amine to form a quaternary ammonium group, and/orreacting with a tertiary phosphine to form a quaternary phosphoniumgroup.

The cation-forming monomers can be stereospecific or non-stereospecific.

In an embodiment, t and t′ of formula (29) are each 1, each R′ at carbon4 is hydrogen, each R′ at carbon 6 is hydrogen, and R″ at carbon 5 isselected from the group consisting of hydrogen, methyl, and ethyl.

Ring opening polymerization of cation-forming monomers of formula (29)produces an initial polycarbonate having a subunit according to formula(30):

wherein

backbone atoms are shown numbered 1 to 6,

L^(a) is a divalent linking group comprising at least 3 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(a), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises at least 1 carbon, and together Q′(R^(a))_(u′) and L^(a)comprise 6 to about 25 carbons,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons,

t is a positive integer having a value of 0 to 2,

t′ is a positive integer having a value of 0 to 2, and

t and t′ cannot both be zero.

More specific cation-forming monomers have the formula (31):

wherein

ring atom 5 is labeled,

L^(b) is a divalent linking group comprising at least 2 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(b), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises at least 1 carbon, and together Q′(R^(a))_(u′) and L^(b)comprise 5 to about 24 carbons, and

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons.

Ring opening polymerization of cation-forming monomers of formula (31)produces a polycarbonate having a subunit according to formula (32):

wherein

ring atom 5 is labeled,

L^(b) is a divalent linking group comprising at least 2 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(b), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises at least 1 carbon, and together Q′(R^(a))_(u′) and L^(b)comprise 5 to about 24 carbons, and

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons.

The cation-forming monomers can have the formula (33):

wherein

ring atom 5 is labeled,

L^(c) is a divalent linking group comprising at least 2 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(c), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises at least 1 carbon, and together Q′(R^(a))_(u′) and L^(c)comprise 5 to about 24 carbons,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl, and

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons.

Ring opening polymerization of cation-forming monomers of formula (33)produces an initial polycarbonate having a subunit according to formula(34):

wherein

ring atom 5 is labeled,

L^(c) is a divalent linking group comprising at least 2 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(c), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3, eachR^(a) is an independent radical having a valency of 1 to 3, wherein eachR^(a) comprises at least 1 carbon, and together Q′(R^(a))_(u′) and L^(c)comprise 5 to about 24 carbons,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl, and

R″ is a monovalent radical selected from the group consisting ofhydrogen, halogens, and alkyl groups comprising 1 to 6 carbons.

Exemplary cation-forming monomers include the cyclic carbonate monomersof Table 1.

TABLE 1

Ring Opening Polymerizations

The reaction mixture used to prepare the disclosed gel-forming blockcopolymers by ring opening polymerization (ROP) comprises avitamin-bearing cyclic carbonate monomer and a dinucleophilicpoly(ethylene oxide) ROP initiator comprising at least one nitrogen. Thereaction mixture can optionally comprise a solvent, ROP catalyst, and/orROP accelerator. Preferably, the ROP is performed by bulk polymerizationof a reaction mixture consisting essentially of the cyclic monomer andpoly(ethylene oxide) initiator at 100-130° C. under vacuum. “Consistingessentially of” means the reaction mixture contains no catalyst,solvent, and accelerator.

The ROP used to prepare the antimicrobial cationic polymers preferablycomprises a solvent, an organocatalyst, a nucleophilic ROP initiator,and one or more cyclic carbonate monomers. A ROP accelerator isoptional.

Using a vitamin-bearing monomer of formula (6) to illustrate a method offorming a gel-forming block copolymer of formula (1) by ring openingpolymerization, a reaction mixture is formed that comprises avitamin-bearing monomer of formula (6) and a di-nucleophilicpoly(ethylene oxide) initiator comprising one or more *—N(H)—* groups,which can be present as a primary amine, secondary amine, tertiaryamine, amide, carbamate, and/or urea group. Heating the reaction mixtureunder vacuum for about 24 hours at a temperature of about 80° C. toabout 150° C. produces a living gel-forming block copolymer of formula(1) having terminal subunits comprising a nucleophilic group capable ofinitiating a ROP. Optionally, the initial gel-forming block copolymer isend capped with a suitable end capping agent.

Using a cation-forming monomer of formula (29) to illustrate a method ofmaking the disclosed cationic polymers, a reaction mixture is formedwhich comprises a cyclic carbonate monomer of formula (29), a catalyst,an optional accelerator, a mono-nucleophilic ROP initiator (optionallycomprising a steroid group), and a solvent. Agitating the reactionmixture at 20-40° C. forms an initial polymer. Optionally the initialpolymer can be endcapped to form an endcapped initial polymer. Theresulting polymer has a structure according to formula (35):

wherein

n′ represents the number of cationic carbonate subunits, wherein n′ hasa value of about 5 to about 45,

Z′ is a monovalent C₁-C₁₅ first end group,

Z″ is a monovalent second end group selected from the group consistingof hydrogen and C₁-C₁₅ moieties,

L^(a) is a divalent linking group comprising at least 3 carbons,

E′ is a substituent capable of reacting to produce a cationic moietyQ′(R^(a))_(u′) linked to L^(a), wherein Q′ is a tetravalentpositive-charged nitrogen or phosphorus, u′ has a value of 1 to 3,

each R^(a) is an independent radical having a valency of 1 to 3, whereineach R^(a) comprises 1 or more carbons, and together Q′(R^(a))_(u′) andL^(a) comprise 6 to about 25 carbons,

each R′ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, methyl, and ethyl,

each R″ is an independent monovalent radical selected from the groupconsisting of hydrogen, halogens, and alkyl groups comprising 1 to 6carbons,

each t is an independent positive integer having a value of 0 to 2,

each t′ is an independent positive integer having a value of 0 to 2, and

no carbonate subunit has t=0 and t′=0.

Z′ can be a residue of the ROP initiator. In an embodiment, Z′ is anS′-L′ group comprising a steroid moiety. In this instance, eachcarbonate subunit of the initial polymer comprises a side chain E′group.

The living end (oxy end) of the initial polymer formed by the ROP has areactive hydroxy group (second end group Z″═H), which is capable ofinitiating another ROP. The living end can be treated with an endcapagent, thereby forming a second end group Z″ that is capable ofpreventing further chain growth and stabilizing the polymer againstunwanted side reactions such as chain scission. The polymerization andendcapping can occur in the same pot without isolating the initialpolymer. Endcap agents include, for example, materials for convertingterminal hydroxy groups to esters, such as carboxylic acid anhydrides,carboxylic acid chlorides, and reactive esters (e.g., p-nitrophenylesters). In an embodiment, the endcap agent is an acylating agent, andthe second end group Z″ is an acyl group. In another embodiment theacylating agent is acetic anhydride, and the second end group Z″ is anacetyl group. In another embodiment, the endcap agent comprises acovalently bound form of a steroid group, a vitamin, or a combinationthereof.

The initial polymer and/or the endcapped initial polymer can be treatedchemically, thermally, and/or photochemically to convert E′ to apositive-charged Q′(R^(a))_(u′) group, thereby forming a cationicpolymer. For example, E′ can be an electrophilic leaving group (e.g.,chloride, bromide, iodide, sulfonate ester, and the like), which iscapable of undergoing a nucleophilic displacement reaction with a Lewisbase (e.g., tertiary amine, trialkyl phosphine) to form a quaternaryammonium group and/or a phosphonium group. In an embodiment, E′ ischloride, bromide, and/or iodide. In another embodiment, the cycliccarbonate monomer is a compound of formula (31) and the initial polymercomprises a subunit of formula (32). In another embodiment, the cycliccarbonate monomer is a compound of formula (33) and the initial polymercomprises a subunit of formula (34).

Also contemplated is a method of forming the cationic polymer using acationic cyclic carbonate monomer that comprises a positive-charged Q′group. In this instance, the ROP forms an initial cationic polymerhaving a living end unit (i.e., a nucleophilic hydroxy end group capableof initiating a subsequent ROP). The living end unit can be endcapped toprevent unwanted side reactions.

Exemplary non-limiting tertiary amines for forming quaternary amines bya nucleophilic substitution reaction with electrophilic E′ groupsinclude trimethylamine, triethylamine, tri-n-propylamine,tri-iso-propylamine, tri-n-butylamine, tri-n-pentylamine,dimethylethylamine, dimethylpropylamine, dimethyl-iso-propylamine,dimethylbutylamine, dimethylpentylamine, dimethylbenzylamine,diethylmethylamine, diethylpentylamine, diethylbutylamine,N,N-dimethylcyclohexylamine, N-methylimidazole, N-ethylimidazole,N-(n-propyl)imidazole, N-isopropylimidazole, N-(n-butyl)imidazole,N,N-diethylcyclohexylamine, N,N-dimethylaniline, N,N-diethylaniline,pyridine, and combinations thereof.

Exemplary non-limiting tertiary phosphines for forming quaternaryphosphonium groups by a nucleophilic substitution reaction withelectrophilic E′ groups include trimethylphosphine, triethylphosphine,tripropylphosphine, tributylphosphine, ethyldimethylphosphine,propyldimethylphosphine, butyldimethylphosphine,pentyldimethylphosphine, hexyldimethylphosphine,heptyldimethylphosphine, octyldimethylphosphine, methyldiethylphosphine,propyldiethylphosphine, butyldiethylphosphine, pentyldiethylphosphine,hexyldiethylphosphine, heptyldiethylphosphine, octyldiethylphosphine,pentyldipropylphosphine, pentyldibutylphosphine,dipentylmethylphosphine, dipentylethylphosphine,dipentylpropylphosphine, dipentylbutylphosphine, tripentylphosphine,hexyldipropylphosphine, hexyldibutylphosphine,cyclohexyl-dimethylphosphine, cyclohexyldiethylphosphine,dihexylmethylphosphine, dihexyl-ethylphosphine, dihexylpropylphosphine,benzyldimethylphosphine, and combinations thereof.

ROP Initiators

Nucleophilic initiators for ROP generally include alcohols, amines,and/or thiols.

Mononucleophilic Initiators

For the above described cationic polymers having one cationic polymerchain (on-armed cationic polymers), the ROP initiator is amono-nucleophilic non-polymeric initiator (e.g., ethanol, n-butanol,benzyl alcohol, and the like). In some instances, the ROP initiator cancomprise a covalently bound form of a biologically active compoundselected from the group consisting of steroids, non-steroid hormones,vitamins, and drugs. For example, mono-nucleophilic ROP initiatorsinclude cholesterol, alpha-tocopherol, and ergocalciferol.

More specific mono-nucleophilic ROP initiators comprise a non-chargedsteroid group S′. The initiator can have a structure according toformula (37):S′-L^(e)  (37),wherein S′ is a steroid group and L is a monovalent group comprising i)1 to about 10 carbons and ii) a nucleophilic initiating group for theROP. Non-limiting examples of ROP initiators of formula (37) includeChol-OPrOH:

and Chol-OTEG-OH:

In the above examples, S′ is a cholesteryl group. Using the preferredmethod of preparing the cationic polymers described below, the S′-L′-*fragment of the cationic polymer is a residue of the ROP initiator whenlinked to the carbonyl end of the polycarbonate backbone. The S′-L′-*fragment derived from Chol-OPrOH has the structure:

The S′-L′-* fragment derived from Chol-OTEG-OH has the structure:

The ROP initiator can be used singularly or in combination with adifferent ROP initiator (e.g., initiators having different steroidgroups and/or different L^(e) groups.) The ROP initiator can bestereospecific or non-stereospecific.

Di-Nucleophilic Initiators for Two-Armed Cationic Polymers

The ROP initiator used to form the above described cationic polymershaving two polymer chains (two-armed cationic polymers) is adi-nucleophilic initiator. Exemplary di-nucleophilic ROP initiatorsinclude ethylene glycol, butanediol, 1,4-benzenedimethanol, and Bn-MPA:

An exemplary di-nucleophilic ROP initiator comprising a steroid group isChol-MPA:

Polyethyleneoxide Initiators for Preparing Gel-Forming Block Copolymers

The ROP initiator used to prepare the gel-forming block copolymers is adi-nucleophilic poly(ethylene oxide) having a number average molecularweight (Mn) of about 5000 to about 25000, and preferably about 10,000 toabout 20,000. The poly(ethylene oxide) initiator comprises at least onenitrogen. The di-nucleophilic poly(ethylene oxide) has independentterminal ROP initiating groups selected from the group consisting ofamines, alcohols, thiols, and combinations thereof. Exemplarynon-limiting di-nucleophilic poly(ethylene oxide) initiators include thefollowing materials.

The polymeric initiators can be used singularly or in combination.

In an embodiment the ROP initiator used to form the gel-forming blockcopolymer is a poly(ethylene glycol) diamine (NH₂—PEG-NH₂), alsoreferred to simply as PEG-diamine.

ROP Solvents

When a solvent is used for the ROP, non-limiting solvents includedichloromethane, chloroform, benzene, toluene, xylene, chlorobenzene,dichlorobenzene, benzotrifluoride, petroleum ether, acetonitrile,pentane, hexane, heptane, 2,2,4-trimethylpentane, cyclohexane, diethylether, t-butyl methyl ether, diisopropyl ether, dioxane,tetrahydrofuran, or a combination comprising one of the foregoingsolvents. A suitable monomer concentration is about 0.1 to 5 moles perliter, and more particularly about 0.2 to 4 moles per liter.

ROP Catalysts

When a catalyst is used for the ROP, less preferred catalysts includemetal oxides such as tetramethoxy zirconium, tetra-iso-propoxyzirconium, tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium,tetra-t-butoxy zirconium, triethoxy aluminum, tri-n-propoxy aluminum,tri-iso-propoxy aluminum, tri-n-butoxy aluminum, tri-iso-butoxyaluminum, tri-sec-butoxy aluminum, mono-sec-butoxy-di-iso-propoxyaluminum, ethyl acetoacetate aluminum diisopropylate, aluminumtris(ethyl acetoacetate), tetraethoxy titanium, tetra-iso-propoxytitanium, tetra-n-propoxy titanium, tetra-n-butoxy titanium,tetra-sec-butoxy titanium, tetra-t-butoxy titanium, tri-iso-propoxygallium, tri-iso-propoxy antimony, tri-iso-butoxy antimony, trimethoxyboron, triethoxy boron, tri-iso-propoxy boron, tri-n-propoxy boron,tri-iso-butoxy boron, tri-n-butoxy boron, tri-sec-butoxy boron,tri-t-butoxy boron, tri-iso-propoxy gallium, tetramethoxy germanium,tetraethoxy germanium, tetra-iso-propoxy germanium, tetra-n-propoxygermanium, tetra-iso-butoxy germanium, tetra-n-butoxy germanium,tetra-sec-butoxy germanium and tetra-t-butoxy germanium; halogenatedcompound such as antimony pentachloride, zinc chloride, lithium bromide,tin(IV) chloride, cadmium chloride and boron trifluoride diethyl ether;alkyl aluminum such as trimethyl aluminum, triethyl aluminum, diethylaluminum chloride, ethyl aluminum dichloride and tri-iso-butyl aluminum;alkyl zinc such as dimethyl zinc, diethyl zinc and diisopropyl zinc;heteropolyacids such as phosphotungstic acid, phosphomolybdic acid,silicotungstic acid and alkali metal salt thereof; zirconium compoundssuch as zirconium acid chloride, zirconium octanoate, zirconiumstearate, and zirconium nitrate.

Preferably, the chemical formula of the catalyst used for the ringopening polymerization does not include an ionic or nonionic form of ametal selected from the group consisting of beryllium, magnesium,calcium, strontium, barium, radium, aluminum, gallium, indium, thallium,germanium, tin, lead, arsenic, antimony, bismuth, tellurium, polonium,and metals of Groups 3 to 12 of the Periodic Table. Metals of Groups 3to 12 of the Periodic Table include scandium, titanium, vanadium,chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium,zirconium, niobium, molybdenum, technetium, ruthenium, rhodium,palladium, silver, cadmium, lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum,tungsten, rhenium, osmium, iridium, platinum, gold, mercury, actinium,thorium, protactinium, uranium, neptunium, plutonium, americium, curium,berkelium, californium, einsteinium, fermium, mendelevium, nobelium,lawrencium, rutherfordium, dubnium, seaborgium, bohrium, hassium,meitnerium, darmstadtium, roentgenium, and copernicium.

Preferred catalysts are organocatalysts whose chemical formulas containnone of the above metals. Examples of organocatalysts for ring openingpolymerizations include tertiary amines such as triallylamine,triethylamine, tri-n-octylamine and benzyldimethylamine4-dimethylaminopyridine, phosphines, N-heterocyclic carbenes (NHC),bifunctional aminothioureas, phosphazenes, amidines, and guanidines.

A more specific organocatalyst isN-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU):

Other ROP organocatalysts comprise at least one1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donatinghydrogen bond catalysts have the formula (38):R²—C(CF₃)₂OH  (38),wherein R² represents a hydrogen or a monovalent radical having 1 to 20carbons, for example an alkyl group, substituted alkyl group, cycloalkylgroup, substituted cycloalkyl group, heterocycloalkyl group, substitutedheterocycloalkyl group, aryl group, substituted aryl group, or acombination thereof. Exemplary singly-donating hydrogen bondingcatalysts are listed in Table 2.

TABLE 2

Doubly-donating hydrogen bonding catalysts have two HFP groups,represented by the formula (39):

wherein R³ is a divalent radical bridging group comprising 1 to 20carbons, such as an alkylene group, a substituted alkylene group, acycloalkylene group, substituted cycloalkylene group, aheterocycloalkylene group, substituted heterocycloalkylene group, anarylene group, a substituted arylene group, and a combination thereof.Representative double hydrogen bonding catalysts of formula (39) includethose listed in Table 3. In a specific embodiment, R² is an arylene orsubstituted arylene group, and the HFP groups occupy positions meta toeach other on the aromatic ring.

TABLE 3

In one embodiment, the catalyst is selected from the group consisting of4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA, 3,5-HFA-St, 1,3-HFAB,1,4-HFAB, and combinations thereof.

Also contemplated are catalysts comprising HFP-containing groups boundto a support. In one embodiment, the support comprises a polymer, acrosslinked polymer bead, an inorganic particle, or a metallic particle.HFP-containing polymers can be formed by known methods including directpolymerization of an HFP-containing monomer (for example, themethacrylate monomer 3,5-HFA-MA or the styryl monomer 3,5-HFA-St).Functional groups in HFP-containing monomers that can undergo directpolymerization (or polymerization with a comonomer) include acrylate,methacrylate, alpha, alpha, alpha-trifluoromethacrylate,alpha-halomethacrylate, acrylamido, methacrylamido, norbornene, vinyl,vinyl ether, and other groups known in the art. Examples of linkinggroups include C₁-C₁₂ alkyl, a C₁-C₁₂ heteroalkyl, ether group,thioether group, amino group, ester group, amide group, or a combinationthereof. Also contemplated are catalysts comprising chargedHFP-containing groups bound by ionic association to oppositely chargedsites on a polymer or a support surface.

The ROP reaction mixture comprises at least one organocatalyst and, whenappropriate, several organocatalysts together. The ROP catalyst is addedin a proportion of 1/20 to 1/40,000 moles relative to the cycliccarbonyl monomers, and preferably in a proportion of 1/1,000 to 1/20,000moles relative to the cyclic carbonyl monomers.

ROP Accelerators.

The ROP polymerization can be conducted with an optional accelerator, inparticular a nitrogen base. Exemplary nitrogen base accelerators arelisted below and include pyridine (Py), N,N-dimethylaminocyclohexane(Me₂NCy), 4-N,N-dimethylaminopyridine (DMAP), trans1,2-bis(dimethylamino)cyclohexane (TMCHD),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (−)-sparteine, (Sp)1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1),1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2),1,3-bis(2,6-di-i-propylphenyl(imidazol-2-ylidene (Im-3),1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4),1,3-di-i-propylimidazol-2-ylidene (Im-5),1,3-di-t-butylimidazol-2-ylidene (Im-6),1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7),1,3-bis(2,6-di-i-propylphenyl)-4, 5-dihydroimidazol-2-ylidene,1,3-bis(2,6-di-i-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8) or acombination thereof, shown in Table 4.

TABLE 4

In an embodiment, the accelerator has two or three nitrogens, eachcapable of participating as a Lewis base, as for example in thestructure (−)-sparteine. Stronger bases generally improve thepolymerization rate.

The catalyst and the accelerator can be the same material. For example,some ring opening polymerizations can be conducted using1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) alone, with no another catalystor accelerator present.

When used, the catalyst is preferably present in an amount of about 0.2to 20 mol %, 0.5 to 10 mol %, 1 to 5 mol %, or 1 to 2.5 mol %, based ontotal moles of cyclic carbonyl monomer.

The nitrogen base accelerator, when used, is preferably present in anamount of 0.1 to 5.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2to 0.5 mol %, based on total moles of cyclic carbonyl monomer. As statedabove, in some instances the catalyst and the nitrogen base acceleratorcan be the same compound, depending on the particular cyclic carbonylmonomer.

The initiator groups are preferably present in an amount of 0.001 to10.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2 to 0.5 mol %,based on total moles of cyclic carbonyl monomer.

In a specific embodiment, the catalyst is present in an amount of about0.2 to 20 mol %, the nitrogen base accelerator is present in an amountof 0.1 to 5.0 mol %, and the nucleophilic initiator groups of theinitiator are present in an amount of 0.1 to 5.0 mol % based on totalmoles of cyclic carbonate monomer.

The catalysts can be removed by selective precipitation or in the caseof the solid supported catalysts, simply by filtration. The catalyst canbe present in an amount of 0 wt % (weight percent) to about 20 wt %,preferably 0 wt % (weight percent) to about 0.5 wt % based on the totalweight of the cationic oligomer and the residual catalyst. The cationicoligomer preferably comprises no residual catalyst.

The ring-opening polymerization can be performed at a temperature thatis about ambient temperature or higher, more specifically 15° C. to 200°C., and even more specifically 20° C. to 80° C. Reaction times vary withsolvent, temperature, agitation rate, pressure, and equipment, but ingeneral the polymerizations are complete within 1 to 100 hours.

When a solvent is present, the ROP polymerization is conducted under aninert (i.e., dry) atmosphere, such as nitrogen or argon, and at apressure of 100 MPa to 500 MPa (1 atm to 5 atm), more typically at apressure of 100 MPa to 200 MPa (1 atm to 2 atm). At the completion ofthe reaction, the solvent can be removed using reduced pressure. Whenthe ROP is performed by bulk polymerization at elevated temperature, theROP is conducted using reduced pressure.

Average Molecular Weight.

The gel-forming block copolymers can have a number average molecularweight (Mn) as determined by size exclusion chromatography of about 5500to about 55000.

The cationic polymers have a number average molecular weight (Mn) asdetermined by size exclusion chromatography of about 1500 to about50,000, more specifically about 1500 to about 30,000. The precursorpolymer to the cationic polymer and/or the cationic polymer preferablycan have a polydispersity index (PDI) of 1.01 to about 2.0, moreparticularly 1.01 to 1.30, and even more particularly 1.01 to 1.25.

In some instances the cationic polymers alone can self-assemble intonanoparticulate micelles in de-ionized water. The cationic polymers canhave a critical micelle concentration (CMC) of about 15 mg/L to about 45mg/L. The micelles can have a minimum inhibitory concentration (MIC) formicrobial growth of about 7 mg/L to about 500 mg/L. In some instances,the MIC is below the CMC, meaning the antimicrobial activity is notdependent on self-assembly of the cationic polymers.

In general, cationic polymers having a DP of 5 to about 45 in whichgreater than 75% of the side chain L^(a)-Q′(R^(a))_(u′) groups of thecationic carbonate subunits contained 8 carbons or less were weaklyactive against Gram-negative and/or Gram-positive microbes and fungi.Moreover, at low DP (<10) the HC50 and/or HC20 values of the cationicpolymers generally fell below 500 mg/L, indicating a trend towardbiocidal properties. Higher HC50 and/or HC20 values (500 mg/L or higher)were generally favored by a DP of about 10 to about 45. The examplesfurther below also show that when at least 25% of the side chain groupsof the cationic carbonate subunits contained 13 or more carbons and theDP was about 10 to about 30, the cationic polymers were highly active(MIC<500 mg/L) against both Gram-negative and Gram-positive microbes andfungi, and had HC50 values of 500 mg/L or higher. Increased inhibitionefficacy and lower red blood cell toxicity (higher HC50 values) wereobtained using a steroid end group Z′. Hemolytic selectivity (HC50/MIC)also rose. The groups Z′, Z″, Z^(c), and C′ can be used to furtheradjust antimicrobial activity, hemolytic selectivity, or to provide asecondary function (e.g., cell recognition capability, enhancement ofcell membrane permeability, and so on).

Moreover, 10 mol % or less of carbonate subunits comprising analpha-tocopheryl (a vitamin E compound) and/or an ergocalciferyl(vitamin D2) side chain moiety was also effective in lowering MIC (i.e.,increasing toxicity to microbes) and/or increasing HC50 values (loweringtoxicity to mammalian red blood cells) when 25% to 100% of the cationiccarbonate subunits comprised 10 to 25 carbons.

Utility

A medical composition comprises a solvent, about 4 wt. % to about 10 wt.% of a disclosed gel-forming block copolymer, and about 0.0001 wt. % toabout 10 wt. % of a drug, wherein weight percent (wt. %) is based ontotal weight of the medical composition. The medical composition has theform of a gel wherein the block copolymer, drug and solvent are bound bynoncovalent interactions. The drug can be a non-polymeric drug or apolymeric drug (e.g., antimicrobial cationic polymer). The polymericdrug can be present in the gel matrix in the form of non-associatedpolymer chains, self-assembled particles (e.g., micelles), and/or othercomplexes formed by noncovalent interactions.

Exemplary non-limiting commercially available drugs include thefollowing, where the generic drug is enclosed in parentheses:13-cis-Retinoic Acid, 2-CdA (Cladribine), 2-Chlorodeoxyadenosine(Cladribine), 5-Azacitidine, 5-Fluorouracil (Fluorouracil), 5-FU(Fluorouracil), 6-Mercaptopurine, 6-MP (6-Mercaptopurine), 6-TG(Thioguanine), 6-Thioguanine (Thioguanine), ABRAXANE® (Paclitaxelprotein bound), ACCUTANE® (Isotretinoin), Actinomycin-D (Dactinomycin),ADRIAMYCIN® (Doxorubicin), ADRUCIL® (Fluorouracil), AFINITOR®(Everolimus), AGRYLIN® (Anagrelide), ALA-CORT® (Hydrocortisone),Aldesleukin, Alemtuzumab, ALIMTA® (Pemetrexed), Alitretinoin(9-cis-retinoic acid), Alkaban-AQ (Vinblastine), ALKERAN® (Melphalan),All-transretinoic Acid (Tretinoin), Alpha Interferon (Interferon Alfa),Altretamine, Amethopterin (Methotrexate), Amifostine, Aminoglutethimide,Anagrelide, ANANDRON® (Nilutamide), Anastrozole, Arabinosylcytosine(Cytarabine), Ara-C(Cytarabine), ARANESP® (Darbepoetin Alfa), AREDIA®(Pamidronate), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane), ARRANON®(Nelarabine), Arsenic Trioxide, Asparaginase, ATRA (All-transretinoicAcid), AVASTIN® (Bevacizumab), Azacitidine, BCG, BCNU (Carmustine),Bendamustine (Bendamustine Hydrochloride), Bevacizumab, Bexarotene,BEXXAR® (Tositumomab), Bicalutamide, BICNU® (Carmustine), BLENOXANE®(Bleomycin), Bleomycin, Bortezomib, Busulfan, BUSULFEX® (Busulfan), C225(Cetuximab), Calcium Leucovorin (Leucovorin), CAMPATH® (Alemtuzumab),CAMPTOSAR® (Irinotecan), Camptothecin-11 (Irinotecan), Capecitabine,CARAC® (Fluorouracil), Carboplatin, Carmustine, Carmustine Wafer,CASODEX® (Bicalutamide), CC-5013 (Lenalidomide), CCI-779 (Temsirolimus),CCNU (Lomustine), CDDP (Cisplatin), CEENU® (Lomustine), CERUBIDINE®(Daunomycin), Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor(Leucovorin), Cladribine, Cortisone (Hydrocortisone), COSMOGEN®(Dactinomycin), CPT-11 (Irinotecan), Cyclophosphamide, CYTADREN®(Aminoglutethimide), Cytarabine, Cytarabine Liposomal, CYTOSAR-U®(Cytarabine), CYTOXAN® (Cyclophosphamide), Dacarbazine, DACOGEN®(Decitabine), Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin,Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal,DAUNOXOME® (Daunorubicin Liposomal), DECADRON™ (Dexamethasone),Decitabine, DELTA-CORTEF® (Prednisolone), DELTASONE® (Prednisone),Denileukin Diftitox, DEPOCYT® (Cytarabine Liposomal), Dexamethasone,Dexamethasone Acetate, Dexamethasone Sodium Phosphate, DEXASONE®(Dexamethasone), Dexrazoxane, DHAD (Mitoxantrone), DIC (Dacarbazine),DIODEX® (Dexamethasone), Docetaxel, DOXIL® (Doxorubicin Liposomal),Doxorubicin, Doxorubicin Liposomal, DROXIA® (Hydroxyurea), DTIC(Dacarbazine), DTIC-DOME® (Decarbazine), Duralone (Methylprednisolone),EFUDEX® (Fluorouracil), ELIGARD® (Leuprolide), ELLENCE® (Epirubicin),ELOXATIN® (Oxaliplatin), ELSPAR® (Asparaginase), EMCYT® (Estramustine),Epirubicin, Epoetin Alfa, ERBITUX® (Cetuximab), Erlotinib, ErwiniaL-asparaginase (Asparaginase), Estramustine, ETHYOL® (Amifostine),ETOPOPHOS® (Etoposide), Etoposide, Etoposide Phosphate, EULEXIN®(Flutamide), Everolimus, EVISTA® (Raloxifene), Exemestane, FARESTON®(Toremifene), FASLODEX® (Fulvestrant), FEMARA® (Letrozole), Filgrastim,Floxuridine, FLUDARA® (Fludarabine), Fludarabine, FLUOROPLE®(Fluorouracil), Fluorouracil, Fluorouracil (cream), Fluoxymesterone,Flutamide, Folinic Acid (Leucovorin), FUDR® (Floxuridine), Fulvestrant,G-CSF (Pegfilgrastim), Gefitinib, Gemcitabine, Gemtuzumab ozogamicin,GEMZAR® (Gemcitabine), GLEEVEC® (Imatinib mesylate), GLIADEL® Wafer(Carmustine Wafer), GM-CSF (Sargramostim), Goserelin, Granulocyte-ColonyStimulating Factor (Pegfilgrastim), Granulocyte Macrophage ColonyStimulating Factor (Sargramostim), HALOTESTIN® (Fluoxymesterone),HERCEPTIN® (Trastuzumab), HEXADROL® (Dexamethasone), HEXALEN®(Altretamine), Hexamethylmelamine (Altretamine), HMM (Altretamine),HYCAMTIN® (Topotecan), HYDREA® (Hydroxyurea), Hydrocort Acetate(Hydrocortisone), Hydrocortisone, Hydrocortisone Sodium Phosphate,Hydrocortisone Sodium Succinate, HYDROCORTONE® Phosphate(Hydrocortisone), Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan(Ibritumomab), IDAMYCIN® (Idarubicin), Idarubicin, IFEX® (Ifosfamide),IFN-alpha (Interferon alfa), Ifosfamide, IL-11 (Oprelvekin), IL-2(Aldesleukin), Imatinib mesylate, Imidazole Carboxamide (Decarbazine),Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2(Aldesleukin), Interleukin-11 (Oprelvekin), INTRON® A (interferonalfa-2b), IRESSA® (Gefitinib), Irinotecan, Isotretinoin, Ixabepilone,IXEMPRA® (Ixabepilone), Kidrolase (Asparaginase), LANACORT®(Hydrocortisone), Lapatinib, L-asparaginase, LCR (Vincristine),Lenalidomide, Letrozole, Leucovorin, LEUKERAN® (Chlorambucil), LEUKINE®(Sargramostim), Leuprolide, Leurocristine (Vincristine), LEUSTATIN®(Cladribine), Liposomal Ara-C, LIQUID PRED® (Prednisone), Lomustine,L-PAM (Melphalen), L-Sarcolysin (Melphalen), LUPRON® (Leuprolide),LUPRON DEPOT® (Leuprolide), MATULANE® (Procarbazine), MAXIDEX®(Dexamethasone), Mechlorethamine, Mechlorethamine Hydrochloride,Medralone (Methylprednisolone), MEDROL® (Methylprednisolone), MEGACE®(Megestrol), Megestrol, Megestrol Acetate (Megastrol), Melphalan,Mercaptopurine (6-Mercaptopurine), Mesna, MESNEX® (Mesna), Methotrexate,Methotrexate Sodium (Methotrexate), Methylprednisolone, METICORTEN®(Prednisone), Mitomycin (Mitomycin C), Mitomycin-C, Mitoxantrone,M-Prednisol (Methylprednisolone), MTC (Mitomycin-C), MTX (Methotrexate),MUSTARGEN® (Mechlorethamine), Mustine (Mechlorethamine), MUTAMYCIN®(Mitomycin-C), MYLERAN® (Busulfan), MYLOCEL® (Hydroxyurea), MYLOTARG®(Gemtuzumab ozogamicin), NAVELBINE® (Vinorelbine), Nelarabine, NEOSAR®(Cyclophosphamide), NEULASTA® (Pegfilgrastim), NEUMEGA® (Oprelvekin),NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib), NILANDRON® (Nilutamide),Nilutamide, NIPENT® (Pentostatin), Nitrogen Mustard (Mechlorethamine),NOLVADEX® (Tamoxifen), NOVANTRONE® (Mitoxantrone), Octreotide,Octreotide acetate (Octreotide), ONCASPAR® (Pegaspargase), ONCOVIN®(Vincristine), ONTAK® (Denileukin Diftitox), ONXOL® (Paclitaxel),Oprelvekin (Interleukin-11), ORAPRED® (Prednisolone), ORASONE®(Prednisone), Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound,Pamidronate, Panitumumab, PANRETIN® (Alitretinoin), PARAPLATIN®(Carboplatin), PEDIAPRED® (Prednisolone), PEG Interferon, Pegaspargase,Pegfilgrastim, PEG-INTRON® (Interferon Alfa-2b), PEG-L-asparaginase,Pemetrexed, Pentostatin, Phenylalanine Mustard (Melphalen), PLATINOL®(Cisplatin), Platinol-AQ (Cisplatin), Prednisolone, Prednisone, PRELONE®(Prednisolone), Procarbazine, PROCRIT® (Epoetin Alfa), PROLEUKIN®(Aldesleukin), Prolifeprospan 20 with Carmustine Implant (CarmustineWafer), PURINETHOL® (6-Mercaptopurine), Raloxifene, REVLIMID®(Lenalidomide), RHEUMATREX® (Methotrexate), RITUXAN® (Rituximab),Rituximab, Roferon-A (Interferon Alfa-2a), RUBEX® (Doxorubicin),Rubidomycin hydrochloride (Daunomycin), SANDOSTATIN® (Octreotide),SANDOSTATIN LAR® (Octreotide), Sargramostim, SOLU-CORTEF®(Hydrocortisone), SOLU-MEDROL® (Methylprednisolone), Sorafenib, SPRYCEL®(Dasatinib), STI-571 (Imatinib Mesylate), Streptozocin, SUl11248(Sunitinib), Sunitinib, SUTENT® (Sunitinib), Tamoxifen, TARCEVA®(Erlotinib), TARGRETIN® (Bexarotene), TAXOL® (Paclitaxel), TAXOTERE®(Docetaxel), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus,Teniposide, TESPA (Thiotepa), Thalidomide, THALOMID® (Thalidomide),THERACYS® (BCG), Thioguanine, Thioguanine Tabloid (Thioguanine),Thiophosphoamide (Thiotepa), THIOPLEX® (Thiotepa), Thiotepa, TICE®(BCG), TOPOSAR® (Etoposide), Topotecan, Toremifene, TORISEL®(Temsirolimus), Tositumomab, Trastuzumab, TREANDA® (BendamustineHydrochloride), Tretinoin, TREXALL® (Methotrexate), TRISENOX® (ArsenicTrioxide), TSPA (Thiotepa), TYKERB® (Lapatinib), VCR (Vincristine),VECTIBIX® (Panitumumab), VELBAN® (Vinblastine), VELCADE® (Bortezomib),VEPESID® (Etoposide), VESANOID® (Tretinoin), VIADUR® (Leuprolide),VIDAZA® (Azacitidine), Vinblastine, Vinblastine Sulfate, VINCASAR PFS®(Vincristine), Vincristine, Vinorelbine, Vinorelbine tartrate(Vinorelbine), VLB (Vinblastine), VM-26 (Teniposide), Vorinostat, VP-16(Etoposide), VUMON® (Teniposide), XELODA® (Capecitabine), ZANOSAR®(Streptozocin), ZEVALIN® (Ibritumomab), ZINECARD® (Dexrazoxane),ZOLADEX® (Goserelin), Zoledronic acid, ZOLINZA® (Vorinostat), andZOMETA® (Zoledronic acid).

Also disclosed is an antimicrobial composition for killing a microbe.The composition comprises water, about 4 wt. % to about 10 wt. % of adisclosed gel-forming block copolymer, and about 0.0001 wt. % to about10 wt. % of an antimicrobial drug, wherein weight percent (wt. %) isbased on total weight of the antimicrobial composition. In anembodiment, the antimicrobial drug is an antimicrobial cationicpolycarbonate (first drug). The composition can further comprise about0.0001 wt. % to about 10 wt. % of an antimicrobial compound (seconddrug), wherein weight percent (wt. %) is based on total weight of theantimicrobial composition. The first drug and the second drug areassociated by noncovalent interactions in the composition. In anembodiment, the antimicrobial compound (second drug) is fluconazole,doxycycline, or a combinations thereof. The examples below show thatthese combinations also can exhibit synergistic enhancement in toxicityto microbes compared to the same composition lacking either theantimicrobial cationic polymer or the antimicrobial compound when testedunder otherwise identical conditions. The antimicrobial aqueous solutioncan be suitable for eradicating a microbial biofilm.

Further disclosed is a method of killing a microbe, comprisingcontacting a microbe with any of the foregoing antimicrobialcompositions.

Exemplary microbes include Staphylococcus epidermidis (S. epidermidis),Staphylococcus aureus (S. aureus), Escherichia coli (E. coli),Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans),Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantEnterococcus (VRE), Acinetobacter baumannii (A. baumannii), Cryptococcusneoformans (C. neoformans), and Klebsiella pneumoniae (K. pneumoniae).

Additionally disclosed is a method of treating a cancer, comprisingperforming a in vivo depot injection of a gel composition near or incontact with a tumor, thereby inhibiting growth of the tumor. The gelcomposition comprises a solvent, a disclosed gel-forming blockcopolymer, and an anti-tumor agent. In an embodiment, the anti-tumoragent is a monoclonal antibody. In another embodiment, the anti-tumoragent is herceptin.

For the examples below, the following definitions are applicable.

HC50 is defined as the concentration (in mg/L) of cationic polymer thatcauses 50% of mammalian red blood cells to undergo hemolysis. HC50values of 500 mg/L or higher are desirable.

HC20 is defined as the concentration (in mg/L) of cationic polymer thatcauses 20% of mammalian red blood cells to undergo hemolysis. HC20values of 500 mg/L or higher are desirable.

Minimum inhibitory concentration (MIC) is defined as the minimumconcentration (in mg/L) of cationic polymer required to inhibit growthof a given microbe for a twenty-four period. An MIC less than 500 mg/Lis desirable. Even more desirable is a MIC of 250 mg/L or less. A lowerMIC indicates higher antimicrobial activity.

Minimum bactericidal concentration (MBC) is defined as the minimumconcentration (in mg/L) of cationic polymer required to kill a givenmicrobe. A lower MBC indicates higher antimicrobial activity.

HC50 selectivity is defined as the ratio of HC50/MIC. An HC50selectivity of 4 or more is desirable. Higher HC50 selectivity valuesindicate more activity against microbial cells and less toxicity tomammalian cells. Likewise, HC20 selectivity is defined as the ratio ofHC20/MIC. An HC20 selectivity of 4 or more is desirable.

The examples further below demonstrate that the gel-forming triblockcopolymers have a large payload-carrying capacity and improved releaseproperties when the linking groups of the gel-forming block copolymercomprise nitrogens capable of donating hydrogen and/or an electron pairto form hydrogen bonds, enabling numerous pharmaceutical-drivenapplications.

EXAMPLES

Materials used in the following examples are listed in Table 5.

TABLE 5 ABBREVI- ATION DESCRIPTION SUPPLIER Alpha-Tocopherol Alfa AesarSodium Nicotinate Alfa Aesar Herceptin (Mw: 145.5 kDa) Roche,Switzerland MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5- Sigma-Aldrich DiphenylTetrazolium Bromide PBS Phosphate-Buffered Saline 1^(st) Base (PBS, pH7.4) KOLLIPHOR RH40; PEG-40 Sigma-Aldrich hydrogenated castor oil;derived from hydrogenated castor oil and ethylene oxide, TMATrimethylamine Sigma-Aldrich MeIm Methyl Imidazole Sigma-Aldrich EtImEthyl Imidazole Sigma-Aldrich Fluc Fluconazole Sigma-Aldrich DXYDoxycycline Sigma-Aldrich HDF Human dermal fibroblasts Sigma-AldrichHO-PEG-OH Poly(ethylene glycol), Mn 10 Sigma-Aldrich kDa or 20 kDaPEG10DA Poly(ethylene glycol) diamine, Rapp Polymere Mn 10790, PDI 1.03;NH₂(CH₂CH₂O)_(n)CH₂CH₂NH₂, n = 243 PEG20DA Poly(ethylene glycol)diamine, Rapp Polymere Mn 22050, PDI 1.06, NH₂(CH₂CH₂O)_(n)CH₂CH₂NH₂, n= 500 Vitamin D2 Ergocalciferol Tokyo Chemical Industry (TCI) VitEα-Tocopherol Alfa Aesar

Herein, Mn is the number average molecular weight, Mw is the weightaverage molecular weight, and MW is the molecular weight of onemolecule.

A p-value is the probability of obtaining a test statistic at least asextreme as the one that was actually observed, assuming that the nullhypothesis is true. The lower the p-value, the less likely the result isif the null hypothesis is true, and consequently the more “significant”the result is, in the sense of statistical significance. The nullhypothesis is oftentimes rejected when the p-value is less than thesignificance level at (Greek alpha), which is often 0.05 or 0.01.P-values are reported below simply as “P”.

Apoptosis refers to the death of cells which occurs as a normal andcontrolled part of an organism's growth or development process.Biochemical events lead to characteristic cell changes (morphology) anddeath. These changes include blebbing, loss of cell membrane asymmetryand attachment, cell shrinkage, nuclear fragmentation, chromatincondensation, and chromosomal DNA fragmentation.

Unless indicated otherwise, materials were purchased from Sigma-Aldrich,TCI or Merck. All solvents were of analytical grade, purchased fromFisher Scientific or J. T. Baker and used as received. Beforetransferring into the glove box, monomers and other reagents (e.g.,initiator, monomer, etc.) were dried extensively by freeze-drying underhigh vacuum.

N-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU) was preparedas reported by R.C. Pratt, et al., Macromolecules, 2006, 39 (23),7863-7871, and dried by stirring in dry THF over CaH₂, filtering, andremoving solvent under vacuum.

1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) was stirred over CaH₂ andvacuum distilled before being transferred to a glove box.

Human dermal fibroblasts were cultured in RPMI1640 medium. All culturemedia were supplemented with 10% fetal calf serum, 100 U/mL penicillinand 100 micrograms/mL streptomycin (HyClone, U.S.A.). MTT was dissolvedin phosphate-buffered saline (PBS, pH 7.4) with a concentration of 5mg/mL, and the solution was filtered with a 0.22 micrometer filter toremove blue formazan crystals prior to use.

Nuclear Magnetic Resonance (NMR) Spectroscopy

The ¹H- and ¹³C-NMR spectra of monomers and polymers were recorded usinga Bruker Avance 400 spectrometer, and operated at 400 and 100 MHzrespectively, with the solvent proton signal as the internal referencestandard.

Molecular Weight Determination by Size Exclusion Chromatography (SEC)

SEC was conducted using tetrahydrofuran (THF) as the eluent formonitoring the polymer conversion and also for the determination ofpolystyrene equivalent molecular weights of the macro-transfer agents.THF-SEC was recorded on a Waters 2695D (Waters Corporation, U.S.A.)Separation Module equipped with an Optilab rEX differentialrefractometer (Wyatt Technology Corporation, U.S.A.) and Waters HR-4E aswell as HR 1 columns (Waters Corporation, U.S.A.). The system wasequilibrated at 30° C. in THF, which served as the polymer solvent andeluent with a flow rate of 1.0 mL/min. Polymer solutions were preparedat a known concentration (ca. 3 mg/mL) and an injection volume of 100microliters was used. Data collection and analysis were performed usingthe Astra software (Wyatt Technology Corporation, U.S.A.; version5.3.4.14). The columns were calibrated with series of polystyrenestandards ranging from Mp=360 Da to Mp=778 kDa (Polymer StandardService, U.S.A.).

Rheological Experiments

Hydrogels and organogels of known concentrations (4 to 8 wt. %) wereprepared by dissolving the copolymers in deionized (DI) water at 25° C.The rheological analysis of the hydrogels was performed on an ARES-G2rheometer (TA Instruments, USA) equipped with a plate-plate geometry of8 mm diameter. Measurements were taken by equilibrating the gels at 25°C. between the plates at a gap of 1.0 mm. The data were collected undercontrolled strain of 0.2% and a frequency scan of 1.0 to 100radians/second. Gelation properties of the polymer suspension wasmonitored by measuring the shear storage modulus (G′) and the lossmodulus (G″) at each point. For shear-thinning studies, the viscosity ofthe hydrogels was monitored as function of shear rate from 0.1 to 10sec⁻¹.

Scanning Electron Microscope (SEM) Imaging of Hydrogel

To minimize morphological perturbations, the hydrogels were cryo-fixedby transferring the sample into a chamber filled with liquid nitrogen. Aday of freeze-drying process was then followed. The morphology of thegel was observed using SEM (Jeol JSM-7400F, Japan).

Preparation of Monomers Preparation of MTC-OH (MW 160.1)

MTC-OH can be prepared by the method of R. C. Pratt, et al., ChemicalCommunications, 2008, 114-116.

Preparation of MTC-C6H5 (MW 326.2)

A 100 mL round bottom flask was charged with bis-MPA, (7), (5.00 g, 37mmol, MW 134.1), bis-(pentafluorophenyl) carbonate (PFC, 31.00 g, 78mmol, MW 394.1), and CsF (2.5 g, 16.4 mmol) rinsed with 70 mLs oftetrahydrofuran (THF). Initially the reaction was heterogeneous, butafter one hour a clear homogeneous solution was formed that was allowedto stir for 20 hours. The solvent was removed in vacuo and the residuewas re-dissolved in methylene chloride. The solution was allowed tostand for approximately 10 minutes, at which time the pentafluorophenolbyproduct precipitated and could be quantitatively recovered. Thispentafluorophenol byproduct showed the characteristic 3 peaks in the ¹⁹FNMR of pentafluorophenol and a single peak in the GCMS with a mass of184. The filtrate was extracted with sodium bicarbonate, water and wasdried with MgSO₄. The solvent was evaporated in vacuo and the productwas recrystallized (ethyl acetate/hexane mixture) to give MTC-C6F5 as awhite crystalline powder. The GCMS had a single peak with mass of 326g/mol. The calculated molecular weight for C₁₂H7F₅O₅ was consistent withthe assigned structure. ¹H-NMR (400 MHz in CDCl₃): delta 4.85 (d, J=10.8Hz, 2H, CH_(a)H_(b)), 4.85 (d, J=10.8 Hz, 2H, CH_(a)H_(b)), 1.55 (s, 3H,CCH₃).

Preparation of MTC-BnCl (MW 298.7)

A flask was charged with MTC-C6F5 (10 g, 30.6 mmol), p-chloromethylbenzyl alcohol (4.8 g, 30.6 mmol), PROTON SPONGE (2 g, 9.3 mmol) and THF(30 mL). The reaction mixture was stirred for 12 hours then addeddirectly to a silica gel column. The product was isolated using diethylether as the eluent to yield 7.45 g (81%) white crystalline powder.

Preparation of MTC-PrCl (MW 236.65)

MTCOH (8.82 g, 55 mmol) was converted to MTCOCl using standardprocedures with oxalyl chloride. In a dry 250 mL round bottom flaskequipped with a stir bar, the formed intermediate was dissolved in 150mL of dry methylene chloride. Under nitrogen flow an addition funnel wasattached in which 3-chloropropanol (4.94 g, 4.36 mL, 52.25 mmol),pyridine (3.95 g, 4.04 mL, 55 mmol), and 50 mL of dry methylene chloridewas charged. The flask was cooled to 0° C. using an ice bath and the topsolution was added drop wise during a period of 30 minutes. The formedsolution was stirred for an additional 30 minutes before the ice bathwas removed and the solution was stirred for an additional 16 hoursunder nitrogen. The crude product MTC-PrCl was directly applied onto asilica gel column and the product was separated by eluting with 100%methylene chloride. The product fractions were removed and the solventwas evaporated, yielding the product as off-white oil, whichcrystallized upon standing. Yield 11 g (85%). ¹H-NMR (CDCl₃) delta: 4.63(d, 2H, CH₂), 4.32 (t, 2H, CH₂), 4.16 (d, 2H, CH₂), 3.55 (t, 2H, CH₂),2.09 (m, 2H, CH₂), 1.25 (s, 3H, CH₃).

Preparation of 5-methyl-5-(3-bromopropyl)oxycarboxyl-1,3-dioxan-2-one,(MTC-PrBr), (MW 281.10)

MTCOPrBr was prepared by the procedure for MTCOPrCl on a 45 mmol scaleusing 3-bromo-1-propanol as the alcohol. The product was purified bycolumn chromatography, and subsequently recrystallized to yield whitecrystals (6.3 g, 49%). ¹H NMR (400 MHz, CDCl₃): delta 4.69 (d, 2H;CH₂OCOO), 4.37 (t, 2H; OCH₂), 4.21 (d, 2H; CH₂OCOO), 3.45 (t, 2H;CH₂Br), 2.23 (m, 2H; CH₂), 1.33 (s, 3H; CH₃). ¹³C NMR (100 MHz, CDCl₃):delta 171.0, 147.3, 72.9, 63.9, 40.2, 31.0, 28.9, 17.3.

Preparation of MTC-VitE Monomer

MTC-OH (3.08 g, 19.3 mmol) was dissolved in anhydrous THF (50 mL) with afew drops of DMF. Oxalyl chloride (3.3 mL, 39.4 mmol) was then addeddropwise and the reaction mixture stirred under a flow of nitrogen for 1hour before volatiles were removed under vacuum. The resultant off-whitesolid was heated to 65° C. for 2-3 minutes to remove any residualreagent and solvent to give the acyl chloride intermediate, MTC-C1. Thesolid was redissolved in dry dichloromethane (50 mL) and chilled to 0°C. using an ice bath. A solution of alpha-tocopherol (8.30 g, 19.3 mmol)and dry triethylamine (3 mL, 21.6 mmol) in dry dichloromethane (50 mL)was subsequently added dropwise over 30 min. The mixture was allowed towarm up to ambient temperature and stirred for an additional 18 hours. Acrude solid was obtained after removal of solvent, and was subjected topurification by column chromatography using silica gel. Hexane wasinitially used as the eluent before gently increasing the polarity tofinally end with 50% ethyl acetate. A second chromatography separationwas carried out using dichloromethane/ethyl acetate (4:1) in order toobtain the desired product in high purity as a white solid (6.05 g,53%). ¹H NMR (400 MHz, CDCl₃): delta 4.92 (d, 2H, J=10.8 Hz, MTC-CH2),4.34 (d, 2H, J=10.8 Hz, MTC-CH2), 2.59 (d, 2H, J=6.7 Hz,tetrahydropyrano-CH2), 2.09 (s, 3H, Ar—CH3), 2.00 (s, 3H, Ar—CH3), 1.96(s, 3H, ArCH3), 1.70-1.90 (m, 2H), 1.00-1.60 (overlapping peaks, 27H),0.80-0.90 (m, 12H, 4×CH3 on hydrophobic tail).

Preparation of MTC-VitD2 Monomer

MTC-VitD2 was prepared according to the general procedure used toprepare MTC-VitE above. ¹H NMR (400 MHz, CDCl₃): δ 6.20 (d, 1H,VitD-C═CH—), 6.01 (d, 1H, VitD-C═CH—), 5.21 (t, 2H, VitD-CH═CH—), 5.08(s, 2H, VitD-C═CH₂), 4.86 (s, 1H, —OCH— of VitD), 4.66 (d, 2H, MTCCH₂—), 4.17 (d, 2H, MTC—CH₂—), 1.15 2.90 (m, 24H, H of VitD andMTC-CH₃), 1.02 (d, 3H, VitD-CH₃), 0.91 (d, 3H, VitD-CH₃), 0.84 (t, 6H,VitD-CH₃), 0.55 (s, 3H, Vit-CH₃).

Preparation of Cationic Polymers Examples 1 to 8

Random cationic copolymers were prepared using MTC-PrBr and MTC-BnClprecursors for the cationic subunits, MTC-VitE as the hydrophobiccomonomer, benzyl alcohol (BnOH) initiator, and DBU/thiourea ascatalysts. Quarternization was performed with trimethylamine. Thereaction sequence is shown in Scheme 1.

The preparation of cationic polymer VE/BnCl(1:30) (i.e., m′:n′ is 1:30)is representative. In a 20 mL vial containing a magnetic stir bar in theglove box, MTC-BnCl (608.8 mg, 2.04 mmol, 30 equiv.), MTC-VitE (40.0 mg,68 micromoles, 1.0 equiv.) and TU (25.2 mg, 68 micromoles, 1.0 equiv.)were dissolved in dichloromethane (3 mL). To this solution, benzylalcohol (BnOH) (7.0 microliters, 68 micromoles, 1.0 equiv.) followed byDBU (10.2 microliters, 68 micromoles, 1.0 equiv.) were added to initiatepolymerization. The reaction mixture was stirred at room temperature for20 minutes and quenched by the addition of excess (˜20 mg) of benzoicacid. The mixture was then precipitated into ice-cold methanol (50 mL)and centrifuged at ˜5° C. for 30 minutes. The resultant semi-transparentoil was dried under vacuum until a foamy white solid was obtained. GPCanalysis of the intermediate was carried out and the polymer was usedwithout further purification. The polymer was subsequently dissolved inacetonitrile, transferred to a Teflon-plug sealable tube and chilled to0° C. Trimethylamine was added to start the quaternization process. Thereaction mixture was stirred at room temperature for 18 hours in thesealed tube. Precipitation of an oily material was observed during thecourse of reaction. The mixture was evacuated to dryness under vacuumand freeze-dried to finally yield a white crisp-foamy solid. The finalpolymer is characterized by 1H NMR to determine the final compositionand purity.

The polymerizations to form the cationic polymers was generallyefficient and gave moderately high yields. In the case of MTC-BnCl, thereaction was quenched within 30 minutes. MTC-PrBr reactions typicallyrequired up to 4 hours. All pre-quaternized polymers were subjected toGPC analysis and all of them were unimodal with PDI<1.3, indicatingwell-controlled polymerization. Proton NMR analysis of the finalquaternized polymers was also consistent with the formulations.

Table 6 lists the cationic random copolymers prepared with MTC-VitE andBnOH initiator, their degree of polymerization (DP), quaternizing agent,CMC, and the total number of carbons of each cationic subunit. For eachpolymer in Table 6, m′=1.

TABLE 6 Feed Monomer 1/ Mole DP^(c) Quaternizing CMC^(b) Ex. CationicPolymer Initiator Endcap^(a) Monomer 2 Ratio (m′:n′) agent (mg/L) 1VE/PrBr(1:15) BnOH None MTC-VitE/ 1:15 1:11 TMA N.D. MTC-PrBr 2VE/PrBr(1:30) BnOH None MTC-VitE/ 1:30 1:20 TMA 105 MTC-PrBr 3VE/PrBr(1:45) BnOH None MTC-VitE/ 1:45 1:35 TMA N.D. MTC-PrBr 4VE/PrBr(1:30)/MeIm BnOH None MTC-VitE/ 1:30 1:24 MeIm N.D. MTC-PrBr 5VE/PrBr(1:30)/EtIm BnOH None MTC-VitE/ 1:30 1:24 EtIm  52 MTC-PrBr 6VE/BnCl(1:10) BnOH None MTC-VitE/ 1:10 1:8  TMA N.D. MTC-BnCl 7VE/BnCl(1:20) BnOH None MTC-VitE/ 1:20 1:16 TMA N.D. MTC-BnCl 8VE/BnCl(1:30) BnOH None MTC-VitE/ 1:30 1:23 TMA N.D. MTC-BnCl ^(a)“None”means the terminal hydroxy group of the polycarbonate chain was notprotected. ^(b)N.D. means not determined. ^(c)Actual, as determined by¹H NMR analysis

Table 7 lists the analytical properties of the cationic randomcopolymers prepared using MTC-VitE.

TABLE 7 Cationic Monomer 1/ Added Actual Ex. Polymer Monomer 2 Selected¹H NMR peaks (intensity) ratio ratio GPC ^(a) 1 VE/PrBr(1:15) MTC-VitE/7.10-7.60 (br, 5H, initiator Ph), 1:15 1:11 1.15 MTC-PrBr 5.15 (br, 2H,initiator CH₂Ph), 4.00-4.50 (m, 66H), 3.55 (m, 22H), 3.30-3.20 (m, 99H,N(CH₃)), 2.10 (m, 22H), 1.00-2.00 (overlapping peaks, VitE), 0.70- 0.90(m, 12H, overlapping CH₃ on VitE) 2 VE/PrBr(1:30) MTC-VitE/ 7.10-7.60(m, 5H, initiator Ph), 1:30 1:20 1.13 MTC-PrBr 5.15 (m, 2H, initiatorCH₂Ph), 4.00-4.50 (m, 120H), 3.46 (m, 40H), 3.30-3.20 (m, 180H, N(CH₃)),2.10 (m, 40H), 1.00-2.00 (overlapping peaks, VitE), 0.70- 0.90 (m, 12H,overlapping CH₃ on VitE) 3 VE/PrBr(1:45) MTC-VitE/ 7.10-7.60 (m, 5H,initiator Ph), 1:45 1:35 1.21 MTC-PrBr 5.15 (m, 2H, initiator CH₂Ph),4.00-4.50 (m, 120H), 3.46 (m, 70H), 3.30-3.20 (m, 315H, N(CH₃)), 2.10(m, 70H), 1.00-2.00 (overlapping peaks, VitE), 0.70- 0.90 (m, 12H,overlapping CH₃ on VitE) 4 VE/PrBr(1:30)/ MTC-VitE/ 9.00-9.50 (m, 24H,C—H Imdz), 1:30 1:24 1.22 MeIm MTC-PrBr 7.50-8.00 (m, 48H, CH=CH Imdz),4.00-4.50 (m, 192H, overlapping peaks), 3.80-4.00 (m, 72H, CH₃ Imdz),2.20 (m, 48H), 1.00-2.00 (overlapping peaks, VitE), 0.80-0.90 (m, 12H,overlapping CH₃ on VitE) 5 VE/PrBr(1:30)/ MTC-VitE/ 9.00-9.50 (m, 24H,C—H Imdz), 1:30 1:24 1.22 EtIm MTC-PrBr 7.70-8.00 (m, 48H, CH=CH Imdz),4.00-4.50 (m, 240H, overlapping peaks), 2.20 (m, 48H), 1.45 (t, 72H, CH₃Imdz), 1.00-2.00 (overlapping peaks, VitE), 0.80- 0.90 (m, 12H,overlapping CH₃ on VitE) 6 VE/BnCl(1:10) MTC-VitE/ 7.10-7.60 (m, 40H,overlapping 1:10 1:8 1.20 MTC-BnCl peaks of initiator Ph and Bn), 5.00-5.40 (m, 18H, overlapping peaks of initiator CH₂Ph and Bn), 4.60 (m,16H), 4.28 (m, 32H), 3.06 (m, 72H, N(CH₃)), 1.00-2.00 (overlappingpeaks, VitE), 0.70- 0.90 (m, 12H, overlapping CH₃ on VitE) 7VE/BnCl(1:20) MTC-VitE/ 7.10-7.70 (m, 69H, overlapping 1:20 1:16 1.20MTC-BnCl peaks of initiator Ph and Bn), 5.00- 5.40 (m, 34H, overlappingpeaks of initiator CH₂Ph and Bn), 4.68 (m, 32H), 4.29 (m, 64H), 3.07 (m,144H, N(CH₃)), 1.00-2.00 (overlapping peaks, VitE), 0.70- 0.90 (m, 12H,overlapping CH₃ on VitE) 8 VE/BnCl(1:30) MTC-VitE/ 7.20-7.70 (m, 97H,overlapping 1:30 1:23 1.21 MTC-BnCl peaks of initiator Ph and Bn), 5.00-5.40 (m, 48H, overlapping peaks of initiator CH₂Ph and Bn), 4.67 (m,46H), 4.30 (m, 92H), 3.07 (m, 207H, N(CH₃)), 1.00-2.00 (overlappingpeaks, VitE), 0.70- 0.90 (m, 12H, overlapping CH₃ on VitE) ^(a)GPC wasperformed on pre-quaternized polymers

Preparation of Triblock Copolymers

The organocatalytic ring opening polymerization (ROP) of MTC-VitE,initiated by poly(ethylene glycol) (HO-PEG-OH) was achieved usingDBU/thiourea catalysts combination according to the Scheme 2.

The ring opening polymerizations (ROP) with MTC-VitE were incomplete andthe conversion efficiency was around 60%. The excess monomers andreagents were removed by repeated precipitation with diethyl ether. Thefinal compositions of the polymers were confirmed by comparison of theOCH₂—CH₂ peak of PEG to the four CH₃ peaks on the MTC-VitE hydrophobictail. The hydrophobicity of the polymer can be adjusted by varying theamount of MTC-VitE moiety. The hydrophilicity can be adjusted by varyingthe HO-PEG-OH length. The triblock copolymers was not endcapped.

Examples 9 to 14

The formation of Example 11, VitE1.25-PEG(20k)-VitE1.25, isrepresentative. In a 20-mL vial containing a magnetic stir bar in theglove box, MTC-VitE (58.9 mg, 100 micromoles, 4.0 equivalent), HO-PEG-OH(Mn=20 kDa, 500 mg, 25 micromoles, 1.0 equivalent) and TU (9.3 mg, 25micromoles, 1.0 equivalent) were dissolved in dichloromethane (4 mL). Tothis solution, DBU (3.7 microliters, 25 micromoles, 1.0 equivalent) wasadded to initiate polymerization. The reaction mixture was allowed tostir at room temperature and aliquots of samples were taken to monitorthe monomer conversion and evolution of molecular weight by ¹H NMRspectroscopy and SEC. After 120 minutes, the reaction mixture wasquenched by the addition of excess (˜20 mg) of benzoic acid and wasprecipitated into ice-cold diethyl ether (2×50 mL). The resultantpolymer was dried in a vial for about 1-2 days until a constant samplemass was obtained, as white powder.

Table 8 summarizes the triblock copolymers formed using the reaction ofScheme 2.

TABLE 8 Mn^(PEG) MTC-VitE DP^(b) Example Name Name 2 (kDa)^(a) FeedRatio (m) f^(PEG)  9 PEG(10k)-4 VitE1.25-PEG(10k)-VitE1.25 10  4 1.2587.2 10 PEG(10k)-8 VitE2.5-PEG(10k)-VitE2.5 10  8 2.5  77.3 11PEG(20k)-4 VitE1.25-PEG(20k)-VitE1.25 20  4 1.25 93.1 12 PEG(20k)-8VitE2.5-PEG(20k)-VitE2.5 20  8 2.5  87.2 13 PEG(20k)-20VitE6.5-PEG(20k)-VitE6.5 20 20 6.5  72.3 14 PEG(20k)-30VitE8.5-PEG(20k)-VitE8.5 20 30 8.5  66.6 ^(a)Number average molecularweight data from supplier ^(b)Based on 1H NMR spectroscopy ^(c) Weightfraction of PEG, f^(PEG) = Mn^(PEG)/(Mn^(PEG) + (2 × DP × 588.83))

Table 9 lists the analysis of the triblock copolymers.

TABLE 9 MTC- VitE Feed Actual Selected ¹H NMR peaks Mole Mole ExampleName Name 2 (intensity) Ratio Ratio GPC 9 PEG(10k)-4 VitE1.25- 3.40-4.00(s, 906H, OCH2CH2 1:4  1:2.5 1.09 PEG(10k)- PEG), 1.00-2.00 (overlappingpeaks, VitE1.25 VitE), 0.75-0.95 (m, 30H, overlapping CH3 on VitE) 10PEG(10k)-8 VitE2.5- 3.40-4.00 (s, 906H, OCH2CH2 1:8  1:5   1.07PEG(10k)- PEG), 1.00-2.00 (overlapping peaks, VitE2.5 VitE), 0.75-0.95(m, 60H, overlapping CH3 on VitE) 11 PEG(20k)-4 VitE1.25- 3.40-4.00 (s,1815H, OCH2CH2 1:4  1:2.5 1.07 PEG(20k)- PEG), 1.00-2.00 (overlappingpeaks, VitE1.25 VitE), 0.75-0.95 (m, 30H, overlapping CH3 on VitE) 12PEG(20k)-8 VitE2.5- 3.40-4.00(s, 1815H, OCH2CH2 1:8  1:5   1.12PEG(20k)- PEG), 1.00-2.00 (overlapping peaks, VitE2.5 VitE), 0.75-0.95(m, 60H, overlapping CH3 on VitE) 13 PEG(20k)-20 VitE6.5- 3.40-4.00 (s,1815H, OCH2CH2 1:20 1:13  1.15 PEG(20k)- PEG), 1.00-2.00 (overlappingpeaks, VitE6.5 VitE), 0.75-0.95 (m, 156H, overlapping CH3 on VitE) 14PEG(20k)-30 VitE8.5- 3.40-4.00(s, 1815H, OCH2CH2 1:30 1:17  1.12PEG(20k)- PEG), 1.00-2.00 (overlapping peaks, VitE8.5 VitE), 0.75-0.95(m, 204H, overlapping CH3 on VitE)

Preparation of Blank Hydrogels Example 15

VitE1.25-PEG(20k)-VitE1.25 (40 mg) was dissolved in HPLC grade water (1mL) at 25° C. to form a 4 wt. % hydrogel.

Example 16

VitE1.25-PEG(20k)-VitE1.25 (80 mg) was dissolved in HPLC grade water (1mL) at 25° C. to form an 8 wt. % hydrogel. The gel was formed in 4hours.

Example 17

VitE2.5-PEG(20k)-VitE2.5 (40 mg) was dissolved in HPLC grade water (1mL) at ambient temperature to form a 4 wt. % hydrogel. The gel wasformed in 4 hours.

Example 18

VitE2.5-PEG(20k)-VitE2.5 (80 mg) was dissolved in HPLC grade water (1mL) at ambient temperature to form a 8 wt. % hydrogel. The gel wasformed in 4 hours.

Preparation of Blank ABA Organogels Example 19

A 10 wt. % organogel was formed by dissolving VitE6.5-PEG(20k)-VitE6.5(20 mg) in KOLLIPHOR RH40 (200 microliters) and heating the mixture at85° C. with stirring at 1400 rpm for 1 hour. HPLC grade water (10microliters) was then added to the organogel with stirring.

Example 20

The procedure of Example 19 was used to prepare a 10 wt. % organogel ofVitE8.5-PEG(20k)-VitE8.5.

Preparation of ABA Copolymer/Sodium Nicotinate Hydrogels Example 21

Sodium nicotinate was dissolved in HPLC grade water at a concentrationof 3 g/L. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) at 25° C. to form a hydrogelcontaining 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt. % sodiumnicotinate based on total weight of the hydrogel.

Example 22

Sodium nicotinate was dissolved in HPLC grade water at a concentrationof 3 g/L. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (80 mg) at 25° C. to form a hydrogelcontaining 8 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt. % sodiumnicotinate based on total weight of the hydrogel.

Example 23

Sodium nicotinate was dissolved in HPLC grade water at a concentrationof 3 g/L. This solution (1 mL) was then added toVitE2.5-PEG(20k)-VitE2.5 (40 mg) at ambient temperature to form ahydrogel containing 4 wt. % VitE2.5-PEG(20k)-VitE2.5 and 0.3 wt. %sodium nicotinate based on total weight of the hydrogel.

Preparation of ABA Copolymer/Herceptin Hydrogels Example 24

The antibody herceptin was dissolved in HPLC grade water at aconcentration of 10 g/L. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) at ambient temperature to form ahydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. %herceptin based on total weight of the hydrogel.

Example 25

The antibody herceptin was dissolved in HPLC grade water at aconcentration of 10 g/L. This solution (1 mL) was then added toVitE2.5-PEG(20k)-VitE2.5 (40 mg) at 25° C., to form a hydrogelcontaining 4 wt. % VitE2.5-PEG(20k)-VitE2.5 and 1.0 wt. % herceptinbased on total weight of the hydrogel.

Preparation of Antimicrobial ABA Triblock/Doxycycline Organogels

To prepare the antimicrobial organogels, the triblock copolymer wasfirst dissolved in KOLLIPHOR RH40 by heating at 85° C. Doxycycline wasseparately dissolved in filtered HPLC water in a cell culture hood. Thetwo solutions were then mixed together at room temperature for theformation of organogels.

Example 26

VitE6.5-PEG(20k)-VitE6.5 (20 mg) was dissolved in KOLLIPHOR RH40 (200microliters), heating at 85° C. and stirring at 1400 rpm for 1 hour.After that, a doxycycline solution (200 g/L in HPLC grade water) (10microliters) was added and stirred to form an organogel containing 10wt. % VitE6.5-PEG(20k)-VitE6.5 and 1 wt. % doxycycline based on totalweight of the hydrogel.

Example 27

VitE8.5-PEG(20k)-VitE8.5 (20 mg) was dissolved in KOLLIPHOR RH40 (200microliters), heating at 85° C. and stirring at 1400 rpm for 1 hour.After that, a doxycycline solution (200 g/L in HPLC grade water) (10microliters) was added and stirred to form an organogel containing 10wt. % VitE8.5-PEG(20k)-VitE8.5 and 1 wt. % doxycycline based on totalweight of the hydrogel.

Preparation of ABA Triblock/Cationic Polymer Hydrogels

To prepare the antimicrobial hydrogels, cationic polymer was firstdissolved in filtered HPLC water at 25° C. in a bio-hood. The resultantsolution was then added to triblock copolymer solid for dissolution andleft to stand at room temperature

Example 28

Cationic polymer VE/BnCl(1:30) (1 mg) was dissolved with sterile HPLCgrade water (1 mL) to form a solution of concentration (1 g/L). Thissolution (1 mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) andallowed to stand 4 hours at ambient temperature to form a cationicpolymer loaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25and 0.1 wt. % VE/BnCl(1:30) based on total weight of the hydrogel.

Example 29

Cationic polymer VE/PrBr(1:30) (1 mg) was dissolved with sterile HPLCgrade water 1 mL to form a solution of concentration (1 g/L). Thissolution (1 mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) andallowed to stand 4 hours at ambient temperature to form a cationicpolymer loaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25and 0.1 wt. % VE/PrBr(1:30) based on total weight of the hydrogel.

Preparation of Two and Three Component Hydrogels Containing FluconazoleExample 30

Fluconazole (0.5 mg) was dissolved in sterile HPLC grade water (1 mL) at25° C. at a concentration of 0.5 g/L. This solution (1 mL) was thenadded to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a fluconazole loadedhydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.05 wt. %fluconazole (500 mg/L) based on total weight of the hydrogel.

Example 31

A solution was prepared containing cationic polymer VE/BnCl(1:30) (0.156mg) (0.5 MBC=156 mg/L for C. albicans) and fluconazole (0.01 mg) insterile HPLC grade water (1 mL) at 25° C. This solution (1 mL) was thenadded to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogelcontaining 4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.0156 wt. %VE/BnCl(1:30) and 0.001 wt. % fluconazole based on total weight of thehydrogel.

Example 32

A solution was prepared containing cationic polymer VE/BnCl(1:30) (0.078mg) at 0.25 MBC (78 mg/L for C. albicans) and fluconazole (0.04 mg) insterile HPLC grade water (1 mL) at 25° C. This solution (1 mL) was thenadded to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogelcontaining 4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.0078 wt. %VE/BnCl(1:30), and 0.004 wt. % fluconazole based on total weight of thehydrogel.

Preparation of Two and Three Component Hydrogels Containing DoxycyclineExample 33

A solution was prepared containing cationic polymer VE/BnCl(1:30)(0.0156 mg) and doxycycline (0.0025 mg) in sterile HPLC grade water (1mL) at 25° C. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogel containing4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.00156 wt. % VE/BnCl(1:30), and0.00025 wt. % fluconazole based on total weight of the hydrogel.

Example 34

A solution was prepared containing cationic polymer VE/BnCl(1:30)(0.0156 mg) and doxycycline (0.005 mg) in sterile HPLC grade water (1mL) at 25° C. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogel containing4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.00156 wt. % VE/BnCl(1:30), and0.0005 wt. % fluconazole based on total weight of the hydrogel.

Example 35

A solution was prepared containing cationic polymer VE/BnCl(1:30)(0.0312 mg) and doxycycline (0.0025 mg) in sterile HPLC grade water (1mL) at 25° C. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogel containing4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.00312 wt. % VE/BnCl(1:30), and0.00025 wt. % fluconazole based on total weight of the hydrogel.

Example 36

A solution was prepared containing cationic polymer VE/BnCl(1:30)(0.0312 mg) and doxycycline (0.0025 mg) in sterile HPLC grade water (1mL) at 25° C. This solution (1 mL) was then added toVitE1.25-PEG(20k)-VitE1.25 (40 mg) to form a loaded hydrogel containing4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.00312 wt. % VE/BnCl(1:30), and0.00025 wt. % fluconazole based on total weight of the hydrogel.Hydrogels prepared at MBC concentration for S. aureus, E. coli, and C.albicans

Example 37

A solution was prepared containing cationic polymer VE/BnCl(1:30) at(0.156 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution (1mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.0156wt. % VE/BnCl(1:30) based on total weight of the hydrogel.

Example 38

A solution was prepared containing cationic polymer VE/PrBr(1:30) at(0.625 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution (1mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.0625wt. % VE/PrBr(1:30) based on total weight of the hydrogel.

Example 39

A solution was prepared containing cationic polymer VE/BnCl(1:30) at(0.3125 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution(1 mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and0.03125 wt. % VE/BnCl(1:30) based on total weight of the hydrogel.

Example 40

A solution was prepared containing cationic polymer VE/PrBr(1:30) at(0.25 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution (1mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.025wt. % VE/PrBr(1:30) based on total weight of the hydrogel.

Example 41

A solution was prepared containing cationic polymer VE/BnCl(1:30) at(0.313 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution (1mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.0313wt. % VE/BnCl(1:30) based on total weight of the hydrogel.

Example 42

A solution was prepared containing cationic polymer VE/PrBr(1:30) at(0.625 mg) in sterile HPLC grade water (1 mL) at 25° C. This solution (1mL) was then added to VitE1.25-PEG(20k)-VitE1.25 (40 mg) to form aloaded hydrogel containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.0625wt. % VE/PrBr(1:30) based on total weight of the hydrogel.

Other Two and Three Component Hydrogels Containing Fluconazole

Example 43

Following the general procedure of Example 30 a fluconazole loadedhydrogel was prepared containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and0.3 wt. % fluconazole (500 mg/L) based on total weight of the hydrogel.

Example 44

Following the general procedure of Example 31 a loaded hydrogel wasprepared containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25, 0.3 wt. %VE/BnCl(1:30) and 0.3 wt. % fluconazole based on total weight of thehydrogel.

Table 10 lists the hydrogel and organogel compositions.

TABLE 10 Component Component Component Component Component Component 1 23 Example 1 2 3 Wt. % Wt. % Wt. % Solvent 15 VitE1.25- 4 Water PEG(20k)-VitE1.25 16 VitE1.25- 8 Water PEG(20k)- VitE1.25 17 VitE2.5- 4 WaterPEG(10k)- VitE2.5 18 VitE2.5- 8 Water PEG(20k)- VitE2.5 19 VitE6.5- 10KOLLIPH PEG(20k)- OR RH40 VitE6.5 20 VitE8.5- 10 KOLLIPH PEG(20k)- ORRH40 VitE8.5 21 VitE1.25- Sodium 4 0.3 Water PEG(20k)- NicotinateVitE1.25 22 VitE1.25- Sodium 8 0.3 Water PEG(20k)- Nicotinate VitE1.2523 VitE2.5- Sodium 4 0.3 Water PEG(20k)- Nicotinate VitE2.5 24 VitE1.25-Herceptin 4 1 Water PEG(20k)- VitE1.25 25 VitE2.5- Herceptin 4 1 WaterPEG(20k)- VitE2.5 26 VitE6.5- Doxycycline 10 1 KOLLIPH PEG(20k)- OR RH40VitE6.5 27 VitE8.5- Doxycycline 10 1 KOLLIPH PEG(20k)- OR RH40 VitE8.528 VitE1.25- VE/BnCl(1:30) 4 0.1 Water PEG(20k)- VitE1.25 29 VitE1.25-VE/PrBr(1:30) 4 0.1 Water PEG(20k)- VitE1.25 30 VitE1.25- Fluconazole 40.05 Water PEG(20k)- VitE1.25 31 VitE1.25- VE/BnCl(1:30) Fluconazole 40.0156 0.001 Water PEG(20k)- VitE1.25 32 VitE1.25- VE/BnCl(1:30)Fluconazole 4 0.0078 0.004 Water PEG(20k)- VitE1.25 33 VitE1.25-VE/BnCl(1:30) Doxycycline 4 0.00156 0.00025 Water PEG(20k)- VitE1.25 34VitE1.25- VE/BnCl(1:30) Doxycycline 4 0.00156 0.0005 Water PEG(20k)-VitE1.25 35 VitE1.25- VE/BnCl(1:30) Doxycycline 4 0.00313 0.00025 WaterPEG(20k)- VitE1.25 36 VitE1.25- VE/BnCl(1:30) Doxycycline 4 0.003130.00025 Water PEG(20k)- VitE1.25 37 VitE1.25- VE/BnCl(1:30) 4 0.0156Water PEG(20k)- VitE1.25 38 VitE1.25- VE/PrBr(1:30) 4 0.0625 WaterPEG(20k)- VitE1.25 39 VitE1.25- VE/BnCl(1:30) 4 0.03125 Water PEG(20k)-VitE1.25 40 VitE1.25- VE/PrBr(1:30) 4 0.25 Water PEG(20k)- VitE1.25 41VitE1.25- VE/BnCl(1:30) 4 0.0313 Water PEG(20k)- VitE1.25 42 VitE1.25-VE/PrBr(1:30) 4 0.0625 Water PEG(20k)- VitE1.25 43 VitE1.25- Fluconazole4 0.3 Water PEG(20k)- VitE1.25 44 VitE1.25- VE/BnCl(1:30) Fluconazole 40.3 0.3 Water PEG(20k)- VitE1.25

Preparation of Herceptin Solutions

For cell culture studies, herceptin was dissolved using sterile HPLCgrade water at the following concentrations, 0.005, 0.02, 0.1, 0.5, 1and 5 g/L.

Preparation of Fluconazole Solutions

For antimicrobial studies, fluconazole was dissolved using sterile HPLCgrade water at the appropriate concentrations. For example, a solutioncontaining 5 mg/L fluconazole was formed by adding sterile HPLC gratewater to a stock solution (1 g/L) of fluconazole.

Rheological Characterization

Rheological analysis of the hydrogels was performed on an ARES-G2rheometer (TA Instruments, USA) equipped with a plate-plate geometry of8 mm diameter. Measurements were taken by equilibrating the hydrogels at25° C. between the plates at a gap of 1.0 mm. The data were collectedunder controlled strain of 0.2% and a frequency scan of 1.0 to 100rad/sec. The shear storage modulus (G′) and loss modulus (G″) ofhydrogels were measured at each point. For shear-thinning studies, theviscosity of the hydrogels was monitored as a function of shear ratefrom 0.1 to 10 sec⁻¹. From the storage modulus G′, the molar weightbetween the effective cross-links, Mc, was calculated using thefollowing equation:G′=ρRT/Mcwhere ρ is the polymer concentration [g/m³], R is the molar gas constantand T is the absolute temperature.

Restoration of elastic modulus of hydrogel after network disruption wasstudied by applying high strain of 100% for 200 seconds and monitoringthe changes in G′ and G″ at a constant frequency of 1 rad/sec.

Scanning Electron Microscope (SEM) Imaging

To minimize morphological perturbations, the hydrogels were cryo-fixedby transferring the sample into a chamber filled with liquid nitrogen. Aday of freeze-drying process was then followed. The morphology of thegel was observed using a scanning electron microscope (SEM) (JeolJSM-7400F, Japan).

In Vitro Release of Sodium Nicotinate from Hydrogel

The release of sodium nicotinate from the hydrogels was studied usingthe dialysis method. Dialysis membrane tube with molecular weight cutoff(MWCO) of 500 Da (Spectrum Laboratories, U.S.A.) containing 0.5 mL gelwas immersed in 25 mL of the release medium (i.e., PBS (pH 7.4)). Thiswas shaken in a water bath at 100 rpm at 37° C. At designated timeintervals, 0.5 mL of the release medium was removed and replaced withfresh medium. The medium that has been removed was mixed with HPLCmobile phase consisting of 50 mM KH₂PO₄ (adjusted to pH 7.0) andmethanol in the volume ratio of 99:1. Drug content was analyzed usinghigh performance liquid chromatography (HPLC, Waters 996 PDA detector,U.S.A.) at 220 nm UV wavelength.

In Vitro Release of Herceptin from Hydrogel

Herceptin-loaded hydrogels were transferred to Transwell inserts(Corning, U.S.A.). The inserts were then immersed in 25 mL of therelease medium (i.e., PBS (pH 7.4)). This was kept shaking in a waterbath at 100 rpm at 37° C. At designated time intervals, 0.1 mL of therelease medium was removed and replaced with fresh medium. The releasedamount of herceptin was quantified using the protein quantification BCAassay (Pierce, U.S.A.).

In Vitro Release of Doxycycline from Organogel

Doxycycline-loaded organogels were transferred to Transwell inserts(Corning, U.S.A.). The inserts were then immersed in 25 mL of therelease medium (either PBS pH 7.4 or 1×10⁴ U/L lipase solution in PBS pH7.4). This was kept shaking in a water bath at 100 rpm at 37° C. Atdesignated time intervals, 1.0 mL of the release medium was removed andreplaced with fresh medium. The release medium was completely exchangedwith fresh medium after each overnight of incubation to maintain theactivities of the lipase. The medium that was collected was mixed withHPLC mobile phase consisting of 25 mM KH₂PO₄:acetonitrile in the volumeratio of 30:70 (adjusted to pH 3.0). Drug content was analyzed usinghigh performance liquid chromatography (HPLC, Waters 996 PDA detector,U.S.A.) at 260 nm UV wavelength.

Cytotoxicity Study of Hydrogel Using MTT Assay

Human dermal fibroblasts were seeded onto 96-well plates at a density of2×104 cells per well, and cultivated in 100 microliters of growthmedium. The plates were then returned to incubators for 24 hours toreach 70% to 80% confluency before treatment. When the desired cellconfluency was reached, the spent growth medium was removed from eachwell and replaced with 50 microliters of fresh medium and 50 microlitersof hydrogel, and incubated for 24 hours. Each condition was tested infour replicates. When the treatment was completed, the culture mediumwas removed and 10 microliters of MTT solution was added with 100microliters of fresh medium. The plates were then returned to theincubator and maintained in 5% CO₂, at 37° C. for a further 3 hours. Thegrowth medium and excess MTT in each well were then removed. 150microliters of DMSO was then added to each well to dissolve theinternalized purple formazan crystals. An aliquot of 100 microliters wastaken from each well and transferred to a new 96-well plate. The plateswere then assayed at 550 nm and reference wavelength of 690 nm using amicroplate reader (Tecan, U.S.A.). The absorbance readings of theformazan crystals were taken to be those at 550 nm subtracted by thoseat 690 nm. The results were expressed as a percentage of the absorbanceof the control cells without any treatment.

Cytotoxicity Study of Organogel Using MTT Assay

Human dermal fibroblasts were seeded onto 24-well plates at a density of6×10⁴ cells per well, and cultivated in 500 microliters of growthmedium. The plates were then returned to incubators for 24 hours toreach 70%-80% confluency before treatment. When the desired cellconfluency was reached, the spent growth medium was removed from eachwell and replaced with 500 microliters of fresh medium. 50 microlitersof organogel was then added into Transwell inserts (Corning, U.S.A.) andimmersed into the culture medium. The cells were then incubated for 24hours with the organogels. Each condition was tested in 3 replicates.When the treatment was completed, the culture medium was removed and 10pl of MTT solution was added with every 100 microliters of fresh medium.The plates were then returned to the incubator and maintained in 5% CO₂at 37° C. for 3 hours. The growth medium and excess MTT in each wellwere then removed. 600 microliters of DMSO was then added to each wellto dissolve the internalised purple formazan crystals. An aliquot of 100microliters was taken from each well and transferred to a new 96-wellplate. The plates were then assayed at 550 nm and reference wavelengthof 690 nm using a microplate reader (Tecan, U.S.A.). The absorbancereadings of the formazan crystals were taken to be those at 550 nmsubtracted by those at 690 nm. The results were expressed as apercentage of the absorbance of the control cells without any treatment.

Mechanical Properties of Hydrogels and Organogels

The influence of concentration, hydrophobic/hydrophilic balance andchemical composition of the amphiphilic copolymers was investigatedusing dynamic mechanical analysis. Polymer concentration stronglyaffects the storage modulus G′ of the hydrogel. FIGS. 1A to 1D aregraphs showing the mechanical properties of blank hydrogels. FIG. 1A isa graph showing the storage (G′) and loss (G″) moduli of blank hydrogelscontaining 4 wt. % and 8 wt. % VitE1.25-PEG(20k)-VitE1.25 in HPLC water(Example 15 and Example 16, respectively). FIG. 1B is a graph showingthe storage (G′) and loss (G″) moduli of blank hydrogels containing 4wt. % and 8 wt. % VitE2.5-PEG(20k)-VitE2.5 in HPLC water (Example 17 andExample 18, respectively). FIG. 1C is a graph showing the viscositydependence on shear rate of blank hydrogels containing 4 wt. % and 8 wt.% VitE1.25-PEG(20k)-VitE1.25 in HPLC water (Example 15 and Example 16,respectively). FIG. 1D is a graph showing the viscosity dependence onshear rate of blank hydrogels containing 4 wt. % and 8 wt. %VitE2.5-PEG(20k)-VitE2.5 in HPLC water (Example 17 and Example 18,respectively).

As shown in FIG. 1A doubling the concentration (4 to 8 wt. %) results in4 to 10 times higher G′ values. In particular, the 8 wt. %VitE2.5-PEG(20k)-VitE2.5 gel has a storage modulus G′ of about 12000 Pa,which is nearly 10-fold greater compared to the 4 wt. % gel (G′ 1400Pa). The influence of the balance between the hydrophobic andhydrophilic portions of the polymers on the gel stiffness can be seen athigher polymer concentration. An increase in the MTC-VitE subunits from1.25 to 2.5 at a concentration of 8 wt. % in HPLC grade water results inan increase in the storage modulus G′ from about 5000 Pa to about 12000Pa. The molecular weight between physical crosslinks Mc was determined,summarized in Table 11. Hydrogels with increasing polymer concentrationdisplayed lower Mc values, which corresponds to lower molecular weightbetween crosslinks and higher crosslink density.

TABLE 11 Gel Mc Example Name Name 2 (wt. %) (kDa) 15 PEG(20k)-4VitE1.25-PEG(20k)-VitE1.25 4 78.6 16 PEG(20k)-4VitE1.25-PEG(20k)-VitE1.25 8 39.6 17 PEG(20k)-8 VitE2.5-PEG(20k)-VitE2.54 71.5 18 PEG(20k)-8 VitE2.5-PEG(20k)-VitE2.5 8 18.2

The hydrogel viscosity dependence on shear rate at 25° C. of FIG. 1C(Examples 15 and 16) and FIG. 1D (Examples 17 and 18) clearlydemonstrate the shear-thinning properties of the gels. Shear thinningresults from disruption of physical crosslinks between the polymerchains with the application of shear stress. Shear-thinning is desirablefor both topical and injectable applications.

FIG. 1E shows the viscosity dependence on shear rate for 10 wt. %organogels prepared with VitE6.5-PEG(20k)-VitE6.5 (Example 19) andVitE8.5-PEG(20k)-VitE8.5 (Example 20) in KOLLIPHOR RH40. The organogelsdisplay high viscosity at low shear rates, indicating a firm,well-bodied structure. At the shear rate increases, the viscosity of thegels falls drastically to become a thin liquid. This indicates theorganogel can be well-spread over the skin for topical delivery oftherapeutic compounds.

In order for hydrogels to be used as an injectable drug depot, it isessential that the low viscosity solution phase rapidly form a gel whenthe shear force terminates. To study this property, a dynamic stepstrain amplitude test (y=0.2 or 100%) was applied on theVitE1.25-PEG(20k)-VitE1.25 (4 wt. %) hydrogel in HPLC grade water(Example 15, labeled −Herceptin in FIG. 2) and the herceptin-loadedhydrogel (Example 24, labeled +Herceptin in FIG. 2. The graph of FIG. 2shows that the initial G′ was about 1400 Pa at a small strain (y=0.2%).When subjected to a high strain (y=100%), the G′ value immediatelydecreased by more than 20 times to ˜67 Pa at 25° C. After 200 seconds ofthe continuous stress, the strain was returned to y=0.2% and the G′ wasimmediately recovered to about 1400 Pa without any loss at 25° C. Thisdynamic step strain test mimics the pushing action during clinicaladministration into the subcutaneous tissue at 25° C. The reversibilityof rheological behavior of the hydrogel is advantageous for use as aninjectable matrix for delivery of therapeutics.

SEM Imaging of Hydrogel

Hydrogels were formed in deionized (DI) water and rapidly transferredinto a chamber filled with liquid nitrogen. The frozen hydrogels werefreeze-dried for a day and then imaged. Scanning electron micrograph(SEM) images of the hydrogel (FIGS. 3A to 3D) shows that thecross-section morphology and porosity of the hydrogel network isstrongly influenced by the polymer concentration,hydrophobic/hydrophilic balance and chemical composition. FIGS. 3A and3B are SEMS of blank hydrogels of VitE1.25-PEG(20k)-VitE1.25 at 4 wt. %(Example 15) and 8 wt. % (Example 16) concentration in HPLC grade water,respectively. FIGS. 3C and 3D are SEMS of blank hydrogels ofVitE2.5-PEG(20k)-VitE2.5 at 4 wt. % (Example 17) and 8 wt. % (Example18) concentration in HPLC grade water, respectively. Long flexiblefibers are present in large proportion in 4 wt. % hydrogels of bothVitE1.25-PEG(20k)—Long flexible fibers are present in large proportionin 4 wt. % hydrogels of both VitE1.25-PEG(20k)—VitE1.25 (FIG. 3A) andVitE2.5-PEG(20k)-VitE2.5 (FIG. 3C), and this is most likely due to theentanglement of the PEG chains. These long flexible fibers vary indiameter from about 0.1 to about 1 micrometer). Nanosized (<1micrometer) spherical structures that occur along the lengths of thefibers, which might be micelles that are formed during the self-assemblyprocess of the polymers. At 8 wt. % concentration the hydrogels appearas nanophase-separated sponge structures (FIGS. 3B and 3D). The porosityof the sponge structures appear to be different at different polymerconcentrations. At 4 wt. %, the hydrogels appear to be more porouscompared to the 8 wt. % hydrogel.

Drug Release from Hydrogel and Organogels

FIG. 4A is a graph plotting the release rate of sodium nicotinate fromloaded hydrogels Example 21 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt. % sodium nicotinate), Example 22(containing 8 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt. % sodiumnicotinate), and Example 23 (containing 4 wt. % VitE2.5-PEG(20k)-VitE2.5and 0.3 wt. % sodium nicotinate). The loaded hydrogels were studied byimmersing the hydrogels in phosphate buffered saline (PBS) (pH 7.4) at37° C. and measuring the concentration of nicotinate in the PBS solutionover time. A higher triblock copolymer concentration results insignificantly slower release of sodium nicotinate (FIG. 4A) from thehydrogel, possibly due to the lower porosity of the gel matrix, whichresults in slower diffusion of the drug molecules through the gel. Thehydrophobic/hydrophilic balance of the copolymers also affects therelease profiles. From FIG. 4A, 4 wt. % of VitE2.5-PEG(20k)-VitE2.5(Example 23) shows faster release of sodium nicotinate compared to 4 wt.% VitE1.25-PEG(20k)-VitE1.25 (Example 21). Without being bound bytheory, this is possibly due to the lower hydrophilicity ofVitE2.5-PEG(20k)-VitE2.5, which might result in a lower extent ofintermolecular hydrogen bonding between the polymer and sodiumnicotinate molecules.

Large biomolecules exhibit a similar trend in release profile. FIG. 4Bis a graph plotting the release rate of herceptin from loaded hydrogelsExample 24 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. %herceptin) and Example 25 (containing 4 wt. % VitE2.5-PEG(20k)-VitE2.5and 1.0 wt. % herceptin). A large difference in herceptin release ratesis observed. Ninety percent of the protein is released in 48 hours from4 wt. % VitE2.5-PEG(20k)-VitE2.5 (Example 25) hydrogel while the sameamount of release from VitE1.25-PEG(20k)-VitE1.25 (Example 24) gel iscompleted in 312 hours.

The release of doxycycline (DXY) was investigated by immersing theorganogels in PBS and measuring the concentration of doxycycline in thePBS solution over time. Lipase was added to the release medium inattempt to accelerate the degradation of the organogels. FIG. 4C is agraph plotting the release rate of doxycycline from organogels Example26 (containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5 and 1.0 wt. %doxycycline) and Example 27 (containing 10 wt. %VitE8.5-PEG(20k)-VitE8.5 and 1.0 wt. % doxycycline), with and withoutlipase. The presence of lipase did not lead to an increase in therelease of doxycycline until hour 77 when close to 20% more antibioticwas released in the enzyme-containing medium withVitE6.5-PEG(20k)-VitE6.5 (Example 26) and VitE8.5-PEG(20k)-VitE8.5(Example 27). In the presence of lipase, degradation of the organogelscould have occurred, resulting in similar amount of antibiotic (˜75%)being released from the two polymers. After 77 hours, multiple peaks ofthe drug showed up on the HPLC chromatogram indicating drug degradation.No further measurements were made.

In Vitro Biocompatibility Study

The hydrogels were assessed for their in vitro biocompatibility byculturing human dermal fibroblast (HDF) cells in the presence ofhydrogels and organogels for 24 hours. FIG. 5 is a bar graph showing thepercentage of viable HDF cells after treatment with blank hydrogelExamples 15 and 16 (containing 4 wt. % and 8 wt. %VitE1.25-PEG(20k)-VitE1.25, respectively) and blank hydrogel Examples 17and 18 (containing 4 wt. % and 8 wt. % VitE2.5-PEG(20k)-VitE2.5). Thehydrogels of lower concentration (4 wt. %) did not exert any toxicity onthe cells. However, cell viability with 8 wt. % ofVitE2.5-PEG(20k)-VitE2.5 hydrogel (Example 18) was about 75%. Withoutbeing bound by theory, this might be due to the high stiffness(G′-12000) and lower porosity of the hydrogel (see FIG. 3D), which leadsto slower diffusion of nutrients to the cells and exclusion ofmetabolites from the cellular environment.

FIG. 6 is a bar graph showing the percentage of viable human dermalfibroblast (HDF) cells after treating the cells with blank organogelExample 19 (containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5), doxycyclineloaded hydrogel Example 26 (containing 10 wt. % VitE6.5-PEG(20k)-VitE6.5and 1 wt. % doxycycline), blank organogel Example 20 (containing 10 wt.% VitE8.5-PEG(20k)-VitE8.5), and doxycycline loaded hydrogel Example 27(containing 10 wt. % VitE8.5-PEG(20k)-VitE8.5 and 1 wt. % doxycycline).

Cytotoxicity of Herceptin Delivered by Hydrogels Against Various CellLines

Herceptin loaded hydrogel Example 24 (4 wt. % inVitE1.25-PEG(20k)-VitE1.25) was tested against HER2/neu-overexpressinghuman breast cancer BT474 cells, low HER2/neu-expressing human breastcancer MCF7 cells and human dermal fibroblasts (HDF) to investigate thetreatment specificity as well as in vitro biocompatibility of thehydrogel. HER2/neu is also known as c-erbB-2.

Human dermal fibroblasts, MCF7 and BT474 cells were seeded onto 24-wellplates at a density of 6×10⁴ cells per well, and cultivated in 500microliters of growth medium. The plates were then returned toincubators for 24 hours to reach ˜70% confluency before treatment. Whenthe desired cell confluency was reached, the spent growth medium wasremoved from each well and replaced with 500 microliters of fresh mediumtogether with 50 microliters of the hydrogel in a Transwell insert andincubated for either 48 or 120 hours. Each condition was tested in fourreplicates. When the treatment was completed, the culture medium wasremoved and 50 microliters of MTT solution was added with 500microliters of fresh medium. The plates were then returned to theincubator and maintained in 5% CO₂, at 37° C., for a further 3 hours.The growth medium and excess MTT in each well were then removed. 600microliters of DMSO was added to each well to dissolve the internalisedpurple formazan crystals. An aliquot of 100 microliters was taken fromeach well and transferred to a new 96-well plate. The plates were thenassayed at 550 nm and reference wavelength of 690 nm using a microplatereader (Tecan, U.S.A.). The absorbance readings of the formazan crystalswere taken to be those at 550 nm subtracted by those at 690 nm. Theresults were expressed as a percentage of the absorbance of the controlcells without any treatment.

FIG. 7 is a bar graph showing the percent of viableHER2/neu-overexpressing human breast cancer BT474 cells as a function ofherceptin concentration after treating the cells with (a) herceptinloaded hydrogel (4 wt. % in VitE1.25-PEG(20k)-VitE1.25) for 48 hours,(b) herceptin solution for 48 hours, (c) herceptin loaded hydrogel (4wt. % in VitE1.25-PEG(20k)-VitE1.25) for 120 hours, (d) and herceptinsolution for 120 hours, each performed using a herceptin concentrationof 0.0005 wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1 wt. %, and 0.5wt. %. The herceptin loaded hydrogels were prepared using the procedureof Example 24. FIG. 7 shows that 48 hour treatment was insufficient forherceptin to exert sufficient killing effect on BT474 cells. About 65%of the BT474 cells still remained viable at even at the highestherceptin concentration tested (i.e., 5 g/L). Interestingly, when thetreatment was extended to 120 hours, the IC50 of herceptin deliveredusing the hydrogel was drastically reduced to 0.02 g/L, whereas theblank hydrogel has minimal cytotoxicity. IC50 is the half maximalinhibitory concentration, and is a quantitative measure of how much of aparticular drug or other substance (inhibitor) is needed to inhibit agiven biological process (or component of a process, i.e., an enzyme,cell, cell receptor or microorganism) by half. The IC50 of the solutionformulation of herceptin was lower, <0.005 g/L. Without being bound bytheory, the higher IC50 value of the hydrogel formulation might be dueto the release kinetics of herceptin from the hydrogel. About 50% of theinitial amount of herceptin was released by 5 days (i.e., 120 hours),and furthermore, the cumulative cytotoxic effect would also be reducedover the 5-day period as compared to the bolus delivery of herceptinsolution. The herceptin-loaded hydrogel was highly effective in killingBT474 cells at high herceptin concentrations (≥1 g/L), where more than70% of the cells were killed in 120 hours. Pharmacokinetic studies haveshown that herceptin has a half-life of 6.2 to 8.3 days, and with thesustained release profile of herceptin from the hydrogel for more than 2weeks, it is anticipated that the hydrogel would be able to deliver acontinuous supply of antibody in vivo and eradicate HER2+ tumors.

FIG. 8 is a bar graph showing the viability of MCF7 cells as a functionof herceptin concentration when treated with (a) herceptin loadedhydrogel (4 wt. % in VitE1.25-PEG(20k)-VitE1.25) for 48 hours, (b)herceptin solution for 48 hours, (c) herceptin loaded hydrogel (4 wt. %in VitE1.25-PEG(20k)-VitE1.25) for 120 hours, (d) and herceptin solutionfor 120 hours, each performed using a herceptin concentration of 0.0005wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1 wt. %, and 0.5 wt. %.Herceptin loaded hydrogels were prepared using the procedure of Example24. With MCF7 cells, herceptin delivered either in the hydrogel orsolution formulation shows negligible cytotoxic effect with more than80% cell viability even after 120 hours treatment at up to herceptinconcentration of 5 g/L. This demonstrates that the herceptin treatmentwas specific towards HER2/neu-overexpressing cancer cells.

FIG. 9 is a bar graph showing the viability of human dermal fibroblast(HDF) cells as a function of herceptin concentration when treated with(a) herceptin loaded hydrogel (4 wt. % in VitE1.25-PEG(20k)-VitE1.25)for 48 hours, (b) herceptin solution for 48 hours, (c) herceptin loadedhydrogel (4 wt. % in VitE1.25-PEG(20k)-VitE1.25) for 120 hours, (d) andherceptin solution for 120 hours, each performed using a herceptinconcentration of 0.0005 wt. %, 0.002 wt. %, 0.01 wt. %, 0.05 wt. %, 0.1wt. %, and 0.5 wt. %. The herceptin loaded hydrogels were prepared usingthe procedure of Example 24. The antibody solution showed slightcytotoxicity against HDF at 5 g/L after 120 hours of treatment. It ispossible that the interaction of herceptin with the epidermal growthfactor receptors (EGFR/ErbB) on the HDR cells causes slight hinderenceto HDF cell proliferation. Herceptin-loaded hydrogel did not showsignificant cytotoxicity at 5 g/L due to the sustained release ofherceptin (only ˜60% herceptin released from the gel after 120 hours).Furthermore, the blank hydrogels showed no cytotoxicity toward HDF evenafter 120 hours of treatment, indicating in vitro biocompatibility.

In Vivo Biocompatibility and Gel Degradation Studies

In order for the vitamin E-functionalized polymeric hydrogel to serve asdrug delivery depot, it is crucial that the hydrogel be biocompatible invivo. To evaluate this property, subcutaneous injection of the blankhydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25) was carried outin mice.

All animal experiments were conducted in accordance with the approvedprotocol from the Institutional Animal Care and Use Committee (IACUC) atthe Biological Resource Centre of Singapore. Female BALB/c mice,weighing 20 g to 25 g were injected subcutaneously with 150 microlitersof blank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25). Atpredetermined periods, mice were sacrificed and the hydrogel and itssurrounding tissue were isolated. For histological examination, sampleswere fixed in 4% neutral buffered formalin and then stained withhematoxylin/eosin (H&E) using standard techniques. To identify theinflammatory cells populated in the subcutaneous tissue and hydrogel,immunohistochemical staining was carried out using a monoclonal ratanti-mouse antibody CD45R (BD Biosciences, U.S.A.) that recognizes theleukocyte common antigen (CD45). The slides were counterstained withhematoxylin/eosin (H&E) to visualize cell nuclei and examined using astereomicroscope (Nikon, U.S.A.).

Histological sections of the hydrogel treated and surrounding micetissue were examined at 1, 2, 4 and 6 weeks post injection (FIG. 10,optical photomicrographs of stained tissue). Within 2 weeks postinjection, the hydrogel remained mostly intact (arrows in FIG. 10) andsome inflammatory cells (indicated by darker DAB stains shown in thedashed circles) had infiltrated into the hydrogel. At 4 weeks, thethickness of the hydrogel had reduced, indicating degradation of thehydrogel. By 6 weeks, the hydrogel had mostly degraded and there was anoticeable reduction in the number of CD45-positive cells in thehydrogel and surrounding tissue. This significant reduction in thenumber of leukocytes (inflammation-mediating cells) indicates that onlymild in vivo tissue response occurred with the administration ofhydrogel and the inflammatory response was only temporary and did notprogress to a chronic phase.

Biodistribution of Herceptin Delivered Using Different Formulations

To evaluate its biodistribution, herceptin was first labeled using ALEXAFLUOR 790 (Invitrogen, U.S.A.). The ALEXA FLUOR dye, with atetrafluorophenyl (TFP) ester moiety, was added to the antibody in amolar ratio of 15:1. The reaction was carried out at room temperaturefor 30 minutes. Purification of the fluorescent conjugate was carriedout via ultracentrifugation. The conjugate was then analyzed using theNANODROP ND-1000 spectrophotometer (NanoDrop Technologies, U.S.A.) andthe degree of labeling was determined to be 1.45 moles ALEXA FLUOR dyeper mole of herceptin.

BT474 tumor-bearing female BALB/c nude mice were used for this study.The mice were divided into 3 groups and administrated with herceptin indifferent formulations: (1) a herceptin loaded hydrogel Example 24(containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 hydrogel and 0.5 wt. %herceptin) (“Herceptin-loaded hydrogel, S.C.”), (2) a herceptin solutioninjected subcutaneously at approximately 1 cm away from the tumor site(“Herceptin solution, S.C.”), and (3) a herceptin solution injectedintravenously (“Herceptin solution, I.V.”). For preparation of (2) and(3) herceptin was dissolved in HPLC grade water at a concentration of 5g/L.

Anesthetic animals were placed on an animal plate heated to 37° C. Thenear-infrared fluorescence was imaged using the ICG filter pairs andexposure time was set to 1 sec. Scans were performed at 1, 2, 3, 6, 8,10 and 13 days post administration. The mice were sacrificed on Day 13and organs involved in drug clearance and metabolism as well as tumortissue were excised and imaged using IVIS (Caliper Life Science,U.S.A.).

FIG. 11 is a series of mouse drawings showing the biodistribution ofALEXA FLUOR 790-labeled herceptin within the mice over a 13 day period.Comparison between the subcutaneous delivery using the hydrogel and theintravenous injection in solution shows that the subcutaneous injectionwas more favorable. The intravenous injection method resulted inherceptin accumulation mainly within organs such as kidneys, liver andlungs, but only a very little amount in the tumor tissue. When herceptinsolution was injected subcutaneously at about 1 cm away from the tumorsite, the antibody accumulated to a greater extent in the tumor comparedto that in the intravenous injection case. This is most likely due tothe proximity to the tumor tissue which allowed accessibility forherceptin to diffuse to the tumor. However, herceptin was still able toenter the circulation system and experience similar biodistribution asthe intravenous administration. On the other hand, the hydrogelformulation provided localized delivery of herceptin, leading to a highamount of herceptin accumulation within the tumor and very little amountin the other organs. The biodistribution patterns of variousformulations will likely influence their anti-tumor efficacy.

In Vivo Anti-Tumor Efficacy

To understand the therapeutic efficacy of herceptin delivery viadifferent routes of administration, herceptin-loaded hydrogel Example 24(4 wt. % in VitE1.25-PEG(20k)-VitE1.25 and 1 wt. % herceptin) injectedsubcutaneously (S.C.) was compared to both intravenous (I.V.) andsubcutaneous administration of herceptin solution in the BT474-tumorbearing mouse model. The mice were divided into 5 groups consisting of:control injected on the first day of treatment (Day 0) with HPLC gradewater (S.C.); herceptin in solution delivered intravenously (I.V.);herceptin in solution delivered subcutaneously (S.C.); blank hydrogel(Example 15) delivered subcutaneously (S.C.) and herceptin-loadedhydrogel (Example 24) delivered subcutaneously (S.C.). Herceptinsolution was prepared by using HPLC grade water to dissolve herceptin at5 g/L. Subcutaneous injections were performed once per mouse at about 1cm away from tumor sites. The administrated dosage of herceptin was 30mg/kg in 150 microliters for all formulations.

Female BALB/c nude mice, weighing 18 g to 22 g were injectedsubcutaneously with 200 microliters of a cell suspension (1:1 withMatrigel) (BD Biosciences, U.S.A.) containing 5×10⁶ BT474 cells. Threeweeks after inoculation (when the tumor volume was 100 mm³ to 120 mm³),the tumor-bearing mice were randomly divided into several groups (7 to10 mice per group).

In a first experiment, Group 1 mice were used as nontreated control,group 2 and 3 mice were given intravenous (30 mg/kg) and subcutaneous(150 microliters) injections of herceptin solution of concentration of1.25 g/L in HPLC grade water, respectively, group 4 and group 5 micewere subcutaneous injections (150 microliters, 30 mg/kg) of blankhydrogel Example 15 (4 wt. % in VitE1.25-PEG(20k)-VitE1.25) andherceptin loaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25and 1.0 wt. % herceptin), respectively, at ˜1 cm away from the tumorsite. All mice were injected only once on the first day of treatment(Day 0).

In a second experiment, Group 1b mice were used as nontreated control,Group 2b and 3b mice were given 4 doses of weekly intravenous andsubcutaneous injections of herceptin (4×10 mg/kg), respectively whilethe Group 4b mice were injected once subcutaneously withherceptin-loaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25and 1.0 wt. % herceptin) at a dosage of 40 mg/kg on the first day oftreatment (Day 0), respectively. For improved clinical relevance, thesubcutaneous injections of herceptin solution and herceptin-loadedhydrogel were carried out at a remote site, ˜4 cm away from the tumor.The tumor size was measured by calipers in two orthogonal diameters andthe volume was calculated as L×W2/2, where L and W are the major andminor diameters respectively. At the end of the treatment, a two-tailedStudent's t test was used to statistically evaluate the difference intumor volume and P<0.05 was considered to indicate a statisticallysignificant difference. In addition, the toxicities of the differentformulations were evaluated by monitoring the change in mouse bodyweight over the course of treatment.

FIG. 12A is a graph showing the change in body weight of BT474-tumorbearing mice after one injection using various herceptin formulations,including blank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25)delivered subcutaneously (“Blank Gel”) herceptin solution deliveredintravenously (“Herceptin Sol (IV, 30 mg/kg, Once”)), herceptin solutiondelivered subcutaneously (“Herceptin Sol (SC, 30 mg/kg, Once”)), andherceptin loaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25and 1.0 wt. % herceptin) delivered subcutaneously (“Herceptin Gel (SC,30 mg/kg, Once”)). The herceptin dosage was 30 mg/kg. No weight loss wasobserved for all mice during the course of treatment, indicating goodtolerance to all treatment conditions.

FIG. 12B is a graph showing change in tumor size of BT474-tumor bearingmice after one injection using various herceptin formulations, includingblank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25) deliveredsubcutaneously (“Blank Gel”) herceptin solution delivered intravenously(“Herceptin Sol (IV, 30 mg/kg, Once”)), herceptin solution deliveredsubcutaneously (“Herceptin Sol (SC, 30 mg/kg, Once”)), and herceptinloaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0wt. % herceptin) delivered subcutaneously (“Herceptin Gel (SC, 30 mg/kg,Once”)). The herceptin dosage was 30 mg/kg. Measurement of tumor sizewith time reveals that the tumor growth inhibition by the solution andhydrogel formulations were different. Mice treated with blank hydrogelExample 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25, “Blank Gel”) had similaraverage tumor volume compared to the control group (P=0.56). Thisindicates that the blank hydrogel Example 15 did not exert any cytotoxiceffect on the tumors. In sharp contrast, the herceptin-loaded hydrogelExample 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. % herceptin,“Herceptin Gel (SC, 30 mg/kg, Once”) reduced the tumor by about 77%(P=0.01) compared to the control group. It is noteworthy that micetreated with the herceptin loaded hydrogel were the only group thatshowed tumor shrinkage. This can be seen by comparing the tumor size onthe initial and final day of treatment course (P<0.001). Furthermore,the anti-tumor efficacy was much more pronounced with the hydrogeltreatment compared to treatment using herceptin solutions deliveredintravenously (“Herceptin Sol (IV, 30 mg/kg, Once”)) or subcutaneously(“Herceptin Sol (SC, 30 mg/kg, Once”)), with P values <0.005. Withoutbeing bound by theory, this is attributed to the herceptin-containinghydrogel having higher localized herceptin concentration at the tumorsite over a longer period of time (as shown in FIG. 11), which enablesherceptin to exert a more cytotoxic effect against the cancer cells.

Histological Analysis

At 28 days post injection of the herceptin formulations, the mice weresacrificed and the tumors as well as normal tissues (heart, lung, liverand kidney) were individually excised and dissected. For histologicalexamination, the samples were fixed in 4% neutral buffered formalin andthen stained with hematoxylin/eosin (H&E) using standard techniques.Apoptotic cells of tumor samples were identified using TerminalTransferase dUTP Nick-End Labeling (TUNEL) assay. The slides werecounterstained with hematoxylin to visualize nuclei and examined using astereomicroscope (Nikon, U.S.A.).

The TUNEL assay reveals apoptotic cells (as a brown3,3′-diaminobenzidine (DAB) stain) by detecting DNA fragmentationresulting from apoptosis. FIG. 13 is a series of photomicrographsshowing tumor cells of BT474-tumor bearing mice after terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at28 days after one injection using various herceptin formulations,including blank hydrogel Example 15 (4 wt. % VitE1.25-PEG(20k)-VitE1.25)delivered subcutaneously (“Blank Gel (S.C.)”), herceptin solutiondelivered intravenously (“Her Sol (I.V.)”), herceptin solution deliveredsubcutaneously (“Her Sol (S.C.”)), and herceptin loaded hydrogel Example24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. % herceptin)delivered subcutaneously (“Her Gel (S.C.”)). The herceptin dosage was 30mg/kg. The scale bar represents 100 micrometers. The color version ofthe photomicrographs of FIG. 13 shows that tumor cells treated withherceptin, regardless of the formulation used, were mostly apoptotic,indicating that anti-tumor mechanism was based on herceptin-inducedapoptosis. H&E staining shows that treatment using herceptin-loadedhydrogel resulted in fewer cells remaining. This further illustrates thehigher anti-tumor efficacy of the subcutaneous deliveredherceptin-loaded hydrogel.

In addition, the anti-tumor efficacy of one-time subcutaneous injectionof herceptin-loaded hydrogel was compared to weekly intravenous andsubcutaneous injections of herceptin solution. Importantly, thesubcutaneous injections were done at a remote site (about 4 cm away fromthe tumor) to increase clinical relevance of the study. The totalherceptin dosage was 40 mg/kg in each group. At the end of thetreatment, a two-tailed Student's t test was used to statisticallyevaluate the differences in tumor volume and P<0.05 was considered toindicate a statistically significant difference.

FIGS. 14A and 14B are graphs showing the changes in tumor size (FIG.14A) and mouse body weight (FIG. 14B) of BT474-tumor bearing mice afterfour weekly administrations of herceptin solution deliveredintravenously (“Herceptin Sol (IV, 4×10 mg/kg, Weekly”) andsubcutaneously (“Herceptin Sol (IV, 4×10 mg/kg, Weekly”), compared toherceptin loaded hydrogel (Example 24) delivered once subcutaneously(“Herceptin Gel (SC, 40 mg/kg, Once”). For the group treated withherceptin solution, the tumor remained similar in size over the 28 dayperiod.

The tumor reduction provided by the subcutaneously deliveredherceptin-loaded hydrogel Example 24 (4 wt. % VitE1.25-PEG(20k)-VitE1.25and 1.0 wt. % herceptin) is significantly greater compared to thesubcutaneously delivered herceptin solution (FIG. 14A). For theherceptin solution group, tumors remained similar in size throughout theentire course of treatment, whereas for the hydrogel treated group, thetumor decreased by 30% (P=0.03) by the end of the treatment. Mouse bodyweight also remained relatively constant for each sample tested (FIG.14B). Without being bound by theory, the superior anti-tumor efficacy ofthe hydrogel formulation is probably due to the protective environmentprovided by the hydrogel network, which greatly reduced the penetrationinto the subcutaneous region of proteolytic enzymes that can degradeherceptin.

During the early phase of treatment, the anti-tumor efficacy of thesubcutaneously injected herceptin-loaded hydrogel Example 24 (4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 1.0 wt. % herceptin) was much morepronounced than the intravenous injected herceptin solution. Thehydrogel-treated group had 61% tumor shrinkage (P<0.001) by Day 3,whereas the solution treated group showed reduction in tumor size onlyafter 2 weeks from commencement of treatment. Interestingly, similar tothe hydrogel-treated group, the mice injected with herceptin solution(intravenous) showed 32% tumor shrinkage (P=0.001) by the end of thetreatment. FIG. 15 is a series of photomicrographs showing tumor cellsof BT474-tumor bearing mice after TUNEL staining at 28 days postinjection of herceptin in solution (intravenous and subcutaneous) andhydrogel (subcutaneous) formulations. The scale bar represents 100micrometers. Herceptin solution delivered intravenously is labeled “HerSol (I.V., weekly”). Herceptin solution delivered subcutaneously islabeled “Her Sol (S.C., weekly”). Herceptin loaded hydrogel (Example 24)delivered subcutaneously is labeled “Her Gel (S.C., one-time”). Theweekly administration of herceptin might allow fresh supply of theantibody to enter the circulation, thereby compensating for the loss ofbioactivity due to proteolysis and degradation in the body system.Therapeutic efficacy of the one-time injection of herceptin-loadedhydrogel was similar to weekly administration of herceptin in solution(intravenous) as the polymer matrix was able to entrap the antibody andrelease it in a sustained manner. Apoptosis and significant reduction inthe number of cancer cells was observed for both treatment conditions.With regards to clinical relevance, the frequency of injections can bedrastically reduced via the use of hydrogel, providing greaterconvenience in administration over the other formulations.

FIG. 16 is a series of photomicrographs of mice heart, lung, liver andkidney cells after H&E staining at 28 days post injection of herceptinsolution formulations (intravenous and subcutaneous) performed on aweekly basis and herceptin loaded hydrogel (subcutaneous) injected onceon the first day of treatment. The scale bar represents 100 micrometers.Pathological analysis of normal tissues (heart, lungs, liver andkidneys) resected from the mice revealed no toxicity.

Antimicrobial Properties

Killing Efficiency

Procedure for hydrogels: E. coli, P. aeruginosa, S. aureus and C.albicans were obtained from ATCC and reconstituted from its lyophilizedform according to the manufacturer's protocol. Bacterial samples werecultured in Tryptic Soy Broth (TSB) solution at 37° C. under constantshaking of 300 rpm. C. albicans was culture in yeast medium at roomtemperature under constant shaking of 70 rpm. Prior to treatment, thebacterial sample was first inoculated and cultured to enter into loggrowth phase. To prepare the antimicrobial hydrogels, cationic polymerwas first dissolved in filtered HPLC water in a bio-hood. The resultantsolution was then added to solid triblock copolymer for dissolution andleft to stand 4 hours at room temperature for the formation of thehydrogels (Example 28 and Example 29). For instance, cationic polymerVE/BnCl(1:30) or cationic polymer VE/PrBr(1:30) was dissolved by usingsterile HPLC grade water to form a polymer solution at variousconcentrations. This solution was then used to dissolveVitE1.25-PEG(20k)-VitE1.25 to form a 4 wt. % hydrogel containing variousconcentrations of the cationic polymers.

For antimicrobial treatment, 50 microliter aliquots of the hydrogelsincorporated with various amounts of cationic polymers were placed intothe wells of a 96-well microplate. The concentration of bacterialsolution was adjusted to give an initial optical density (O.D.) readingof approximately 0.07 at 600 nm wavelength on a microplate reader(TECAN, Switzerland), which corresponds to the concentration ofMcFarland 1 solution (3×10⁸ CFU/mL). The bacterial solution was thendiluted and an equal volume of bacterial suspension (3×10⁵) was addedinto each well. The 96-well plate was kept in an incubator at 37° C.under constant shaking of 300 rpm for 18 hours for E. coli, P.aeruginosa, S. aureus and C. albicans. After treatment, the samples weretaken for a series of tenfold dilution, and plated onto agar plates. Theplates were incubated for 24 hours at 37° C. and the number ofcolony-forming units (CFU) was counted. Bacteria treated in hydrogelwithout cationic polycarbonates were used as negative control, and eachtest was carried out in 3 replicates. Minimum bactericidal concentration(MBC) is defined herein as the lowest concentration of the antimicrobialcomposition that eliminates >99.9% of the microbes.

Procedure for organogels: E. coli was obtained from ATCC andreconstituted from its lyophilized form according to the manufacturer'sprotocol. Bacterial samples were cultured in Tryptic Soy Broth (TSB)solution at 37° C. under constant shaking of 300 rpm. Prior totreatment, the bacterial sample was first inoculated and cultured toenter into log growth phase. For antimicrobial treatment, 20 microlitersof the organogels (Example 26 and Example 27) containing 1 wt. %doxycycline was added to sterile vials. The concentration of bacterialsolution was adjusted to give an initial optical density (O.D.) readingof approximately 0.07 at 600 nm wavelength on a microplate reader(TECAN, Switzerland), which corresponds to the concentration ofMcFarland 1 solution (3×10⁸ CFU/mL). The bacterial solution was thendiluted and an equal volume of bacterial suspension (3×10⁵) was addedinto each vial and incubated at 37° C. under constant shaking of 300 rpmfor 18 hours. After treatment, the samples were taken for a series oftenfold dilution, and plated onto agar plates. The plates were incubatedfor 24 hours at 37° C. and the number of colony forming units (CFU) wascounted. Bacteria treated with KOLLIPHOR RH40 were used as negativecontrol, and each test was carried out in 3 replicates.

Analysis of Drug Interactions

To assess the antimicrobial effects of the drug combinations (e.g.,cationic polymer/doxycylcine and/or cationic polymer/fluconazole) thecheckerboard and isobologram method of analyzing drug interactions wereused. For the checkerboard method, the fractional inhibitoryconcentration (FBC) was calculated for each component in eachcombination dose. The types and extent of interaction was determined bycalculating the FBC index, which is the ratio of the MBC of a drug incombination and MBC of the drug alone. For two interacting drugs, A andB, the sum of the FBCs indicates the extent of the interaction. Synergyis defined as ΣFIC index ≤0.5. Indifference was defined as ΣFIC indexof >0.5 but ≤4 and antagonism as a ΣFIC index of >4.0. As for theisobologram method, evaluation of drug interaction is performed at theMBC level. Using graphical analysis, the concentrations required toproduce the effect of >99.9% killing efficiency are determined for eachcomponent and plotted on the x and y axes of a two-coordinate plot. Aline is drawn to connect these two points and this is defined as theline of additivity. Thereafter, treatment is performed with the drugs incombination at varying concentrations. The concentrations of fluconazoleand cationic polymer in the combination that provide the same effect areplaced in the same plot. Effect of the drug interaction is determinedaccording to the position of the points with respect to the line ofadditivity. Synergy, additivity, or antagonism is represented when thepoint is located below, on, or above the line, respectively.

Biofilm Formation and Treatment

S. aureus and E. coli were grown overnight in tryptic soy broth (TSB) at37° C. and diluted in TSB to 3×10⁶ and 3×10⁸ CFU/mL before use. C.albicans was grown overnight in yeast medium at room temperature anddiluted to 3×105 CFU/mL before use. 100 microliters of the diluted cellsuspension were then inoculated into each well of 96-well plate andcultured for 7 to 10 days depending on their growth rates. Due todifferences in the rate of biofilm formation, S. aureus and C. albicanswere kept shaking at 100 rpm, 37° C. and 50 rpm, 25° C., respectively.E. coli was incubated at non-shaking conditions at 37° C. The culturemedium was changed every day with PBS being added to wash off theplanktonic and loosely adhered cells before replacing with fresh medium.Treatment was carried out by first removing the spent medium. Thebiofilm was washed gently with PBS to remove the planktonic and looselyadhered cells. The biofilm was then incubated with 50 microliters ofhydrogel composition for 24 hours.

Biomass Assay

The biomass left after treatment was analyzed using crystal violet (CV)staining assay. The spent medium and hydrogel was gently removed and thebiofilm was gently washed with PBS to remove the planktonic cells.Fixation was carried out by adding 100 microliters of methanol to thebiofilm and removed after 15 min. Following this, 100 microliters ofcrystal violet staining (0.1 w/v %) was added to the fixed biofilm andincubated for 10 minutes. Excess crystal violet was washed offthoroughly using HPLC water. The remaining crystal violet bound to thebiofilm was extracted using 200 microliters of 33% glacial acetic acid.An aliquot of 150 microliters was then taken from each well andtransferred to a fresh 96-well plate. The absorbance was then measuredat 570 nm using a microplate reader (Tecan, Switzerland).

XTT Reduction Assay

XTT assay was used for quantifying viable cells in the biofilms afterhydrogel treatment by measuring the mitochondrial enzyme activity of thecells. It is based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT) in the metabolically active microbial cells to a watersoluble formazan. XTT solution (1 mg/mL) and menadione solution (0.4 mM)were individually prepared by dissolution in deionized water. Rightbefore the assay, the two components were mixed together at a volumeratio of XTT:menadione 5:1. During the assay, medium was first removedand biofilm were carefully washed with PBS to remove the planktoniccells. 120 microliters of PBS and 14.4 microliters of the XTT-menadionemixture was then added to each well and incubated for 3 hours. Analiquot of 100 microliters was then taken from each well and transferredto a fresh 96-well plate. The absorbance was then measured at 490 nmusing a microplate reader (Tecan, Switzerland).

Field Emission-Scanning Electron Microscopy (FE-SEM)

After treatment, the biofilm was gently washed with PBS and fixed with4% formaldehyde for 30 minutes. Next, the biofilm was washed with DIwater to remove the formaldehyde and a series of ethanol washes (35, 50,75, 90, 95 and 100%) was carried for dehydration of the samples. Aftertwo days of air-drying the samples were mounted on carbon tape andcoated with platinum for SEM analysis under a field emission scanningelectron microscope (JEOL JSM-7400F, Japan).

Mechanical Properties of Triblock Copolymer/Cationic Polymer Hydrogels

The effects of cationic polymers on the mechanical properties ofhydrogel matrix were investigated. Cationic polymer (0.1 wt. %;equivalent to 1000 mg/L) was added during the formation of 4 wt. %VitE1.25-PEG(20k)-VitE1.25 hydrogel in HPLC water. FIG. 17A is a bargraph of G′ values of cationic polymer loaded hydrogels Example 28(containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and 0.1 wt. % cationicpolymer VE/BnCl(1:30) in HPLC water) and Example 29 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.1 wt. % cationic polymer VE/PrBr(1:30)in HPLC water). Also shown is blank hydrogel Example 15 (containing 4wt. % VitE1.25-PEG(20k)-VitE1.25). As shown in FIG. 17A, the G′ valuesare in a range of 1400 Pa to 1600 Pa. There is little difference instiffness between the blank hydrogel and the cationic polymer loadedhydrogels.

FIG. 17B is a graph of viscosity versus shear rate profile of thecationic loaded hydrogels of FIG. 17A. The cationic polymer loadedhydrogels (Example 28 and Example 29) display high viscosity at lowshear rates, indicating a firm, well-bodied structure. At the shear rateincreases, the viscosity of the gels falls drastically to become a thinliquid. This shear-thinning profile of the hydrogels results from thedisruption of physical cross-links between the polymer chains with theapplication of shear stress. Consequently, this indicates that they canbe well-spread over the skin for topical treatment of dermal infection.

Antimicrobial Activity Studies of ABA Triblock/Cationic PolymersDelivered Using Hydrogel

Antimicrobial activities of two cationic polymer loaded hydrogels wereevaluated against S. aureus, E. coli and C. albicans as representativemodels of Gram-positive, Gram-negative bacteria and fungus respectively.These microbes are common pathogens that often manifest on dermalwounds, and can be treated via topical delivery of antibiotics toinfected areas.

VE/BnCl(1:30) and VE/PrBr(1:30) were loaded into 4 wt. %VitE1.25-PEG(20k)-VitE1.25 hydrogels (Example 28 and Example 29,respectively). Due to the disparity in antimicrobial efficacies of thetwo cationic polymers, different concentration ranges were used for thepreparation of the hydrogels. The hydrogels were challenged with aninoculum of 3×10⁵ CFU/mL and proliferation capacity of the survivedcells was then assessed 24 hours later via the spread plate technique.This method of examining the antimicrobial activity is akin to measuringthe minimum inhibitory concentration (MIC) of the cationic polymers insolution.

FIGS. 18A to 18C are bar graphs showing the killing efficiency againstStaphylococcus aureus (S. aureus), Escherichia coli (E. coli), andCandida albicans (C. albicans), respectively, of cationic polymer loadedhydrogels containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25 and variousconcentrations of cationic polymers VE/BnCl(1:30) or VE/PrBr(1:30). Theconcentration indicated on the horizontal axis refers to cationicpolymer. The results show that the cationic polymers delivered byhydrogel are broadly antimicrobial against bacteria and fungi.

Table 12 lists the minimum bactericidal concentrations (MBC) of thecationic polymer with and without triblock copolymer against S. aureus,E. coli, and C. albicans. The MBC value refers to the amount of cationicpolymer in mg/L. For the cationic polymer alone (Examples 2 and 8), thesolution MBC value is equal to the solution minimum inhibitoryconcentration (MIC) against each microbe.

TABLE 12 MBC^(a) Wt. % Wt. % (mg/L) Triblock Cationic Triblock CationicS. E. C. Example Copolymer Polymer copolymer Polymer aureus colialbicans  2 VE/PrBr(1:30)  63^(b)  250^(b) 250^(b)  8 VE/BnCl(1:30) 31^(b)  31^(b) 250^(b) 28 VitE1.25- VE/BnCl(1:30) 4 0.1 156.2  312.5312.5 PEG(20k)- VitE1.25 29 VitE1.25- VE/PrBr(1:30) 4 0.1 312.5 2500.0625.0 PEG(20k)- VitE1.25 ^(a)MBC values refer to the amount of cationicpolymer in mg/L ^(b)MBC = MIC

Table 12 shows the presence of the triblock copolymer lowers theantimicrobial efficacy of the cationic polymer (i.e., increases the MBCrelative to the cationic polymer alone). The loaded hydrogels are mosteffective in inhibiting Gram-positive S. aureus proliferation. S. aureusrequires the lowest amount of each cationic polymer (MBC=156.2 mg/L forVE/BnCl(1:30) and 312.5 mg/L for VE/PrBr(1:30)). The cationic polymerloaded hydrogels were less able to inhibit the proliferation ofGram-negative E. coli (MBC=312.5 mg/L for VE/BnCl(1:30) and 2500 mg/Land VE/PrBr(1:30)). The antimicrobial effect on C. albicans (fungus) wasintermediate to that of the two bacteria species (MBC=312.5 mg/L forVE-BnCl(1:30) and 625 mg/L for VE/PrBr(1:30)). The MBC value of thecationic polymer (Table 12) is lower than the concentrations at whichthe cationic polymers become hemolytic towards mammalian cells.

The loaded hydrogels are consistent with the solution properties of thecationic polymers alone. That is, the cationic polymer VE/BnCl(1:30)exhibits greater antimicrobial/antifungal properties compared toVE/PrBr(1:30) whether delivered in solution or as a hydrogel complex.

Antimicrobial Activity of Doxycycline-Loaded Organogels

The antimicrobial effectiveness of drug-loaded organogels (Examples 26and 27) was tested on E. coli. E. coli strains from skin and soft tissueinfections can exhibit strong virulence potential. FIG. 19 is a bargraph showing the number of viable bacterial colony-forming units (CFU)after 18 hour treatment of E. coli. with blank organogels (Examples 19and 20, labeled “6.5VE-PEG20k-6.5VE” and “8.5VE-PEG20k-8.5VE”,respectively) and doxycycline loaded organogels (Examples 26 and 27,labeled “6.5VE-PEG20k-6.5VE 1 wt. % DXY” and “8.5VE-PEG20k-8.5VE 1 wt. %DXY”, respectively). The doxycycline loaded organogels demonstrated 100%bactericidal activity (0 CFU). Although the doxycycline concentrationused is higher than the reported minimum bactericidal concentration(MBC) range in several E. coli strains, 1 wt. % was selected becausethis is a typical loading content in clinically-approved doxycyclineformulations such as NanoDOX® and ATRIDOX®.

Synergism of Fluconazole and Cationic Polymer VE/PrBr(1:15) Delivered bySolution

The following demonstrate synergistic enhancement of antimicrobialactivity using cationic polymer VE/PrBr(1:15) in combination withfluconazole delivered via solution. Fluconazole (Fluc) is a member ofthe azole family of antifungal agents that possess good activity againstC. albicans and exhibits low toxicity. While being considerably safe forclinical applications, the downside of using azoles is that they areonly fungistatic, not fungicidal.

A stock solution of cationic polymer VE/PrBr(1:15) (5 mg) in sterileHPLC grade water (10 mL) was prepared having a final concentration of500 mg/L (500 ppm). A second stock solution of fluconazole (1 mg) insterile HPLC grade water (100 mL) was prepared having a finalconcentration of 10 mg/L (10 ppm). Three solutions were prepared: (1)cationic polymer alone at 1.0MIC=250 mg/L (250 ppm) against C. albicans,(2) A cationic polymer/fluconazole solution containing VE/PrBr(1:15) at0.5MIC=125 mg/L (125 ppm) against C. albicans and fluconazole at 2.5mg/L (2.5 ppm), and (3) fluconazole alone at 5.0 mg/L (5 ppm).Formulation (2) was obtained by combining 125 microliters each of thestock solutions of cationic polymer and fluconazole and diluting theresulting solution with 750 microliters of sterile HPLC grade water.

FIG. 20 is a bar graph showing the killing efficiencies of the threesolutions against C. albicans. The cationic polymer solution (1)achieved 99.98% killing efficiency at 1.0MIC=250 mg/L (250 ppm). Thefluconazole solution (3) achieved 93.53% killing efficiency at 5.0 mg/L(5 ppm). However, solution (2) containing VE/PrBr(1:15) and fluconazoleachieved 100% killing efficiency using VE/PrBr(1:15) at 0.5MIC=125 mg/L(125 ppm) and fluconazole at 2.5 mg/L (2.5 ppm).

FIG. 21 is a graph (isobologram) demonstrating the synergy of theVE/PrBr(1:15)/fluconazole combination compared to VE/PrBr(1:15) aloneand fluconazole alone delivered by solution against C. albicans. Thesynergy is indicated by the drug combination dose that lies to the leftof the line of additivity, shown as a square inside the triangle.

Synergism of Fluconazole and Cationic Polymer VE/BnCl(1:30) Delivered bya Hydrogel

The following results demonstrate synergistic enhancement ofantimicrobial activity against C. albicans using three componenthydrogel containing VitE1.25-PEG(20k)-VitE1.25, cationic polymerVE/BnCl(1:30) and fluconazole. Three hydrogels were compared: (1)fluconazole loaded hydrogel Example 30 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.05 wt. % fluconazole) used at a loadedhydrogel concentration of 500 mg/L, (2) cationic polymer/fluconazoleloaded hydrogel Example 31 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25, 0.0156 wt. % cationic polymer VE/BnCl(1:30),and 0.001 wt. % fluconazole) used at a concentration of fluconazole=10mg/L and VE/BnCl(1:30)=156 mg/L (0.5MBC), and (3) cationicpolymer/fluconazole loaded hydrogel Example 32 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25, 0.0078 wt. % cationic polymer VE/BnCl(1:30),and 0.004 wt. % fluconazole) used at a concentration of fluconazole=40mg/L and VE/BnCl(1:30)=78 mg/L (0.25MBC). FIG. 22 is a bar graphcomparing the killing efficiency against C. albicans of the threehydrogels. Even at a high concentration of 500 mg/L, fluconazole alonewas able to kill only 99.56% of C. albicans, whereas the drugcombination of Example 31 used at a concentration of fluconazole=10 mg/Land VE/BnCl(1:30)=156 mg/L (0.5MBC) killed 99.99% of C. albicans (FIG.22, middle bar).

FIG. 23 is a graph showing the release rate of fluconazole fromfluconazole loaded hydrogel Example 43 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.3 wt. % fluconazole, upper curve), andcationic polymer/fluconazole loaded hydrogel Example 44 (containing 4wt. % VitE1.25-PEG(20k)-VitE1.25), 0.3 wt. % cationic polymerVE/BnCl(1:30), and 0.3 wt. % fluconazole, lower curve). The results showthat about 80% of the fluconazole was released within 4 hours from thetwo component hydrogel Example 43 (FIG. 23, upper curve), and about 40%of the fluconazole was released within about 7 hours from the threecomponent hydrogel Example 44 (FIG. 23, upper curve).

By combining this fungistatic drug and cationic polymer in a hydrogelmatrix, significant improvement in the therapeutic efficacy at twocombination doses was observed. The FBC index of the combination doseswas ˜0.5 and ˜0.25 respectively, indicating synergistic interaction fromthe codelivery of the two compounds. Furthermore, the isobologram methodof analyzing drug interactions further illustrates the strong synergismbetween fluconazole and VE/BnCl(1:30) when delivered by the hydrogel.FIG. 24 is an isobologram demonstrating the synergy of the combinationdosages (VE/BnCl(1:30)/fluconazole) for minimum bactericidal activitywhen delivered by loaded hydrogel Example 31 (4 wt. %VitE1.25-PEG(20k)-VitE1.25), 0.0156 wt. % cationic polymerVE/BnCl(1:30), and 0.001 wt. % fluconazole) and loaded hydrogel Example32 (4 wt. % VitE1.25-PEG(20k)-VitE1.25), 0.0078 wt. % cationic polymerVE/BnCl(1:30), and 0.004 wt. % fluconazole). Synergy between thecationic polymer and fluconazole is shown by the drug combination dosagelying to the left of the line of additivity for each loaded hydrogel,shown as a square inside the triangle. The upper square corresponds toExample 31, the bottom square corresponds to Example 32.

Synergism of Doxycycline (DXY)/Cationic Polymer Delivered by a Hydrogel

DXY is a tetracycline antibiotic. Loaded hydrogels were prepared withVitE1.25-PEG(20k)-VitE1.25, DXY, and cationic polymer VE/BnCl(1:30). Thehydrogels were tested against Pseudomonas aeruginosa (P. aeruginosa), acommon pathogen in hospital patients with greater than 1 week stays. TheMBC of DXY against P. aeruginosa is about 20-30 mg/L (30 ppm). The MBCof VE/BnCl(1:30) against P. aeruginosa is about 500 mg/L (500 ppm). Fourhydrogels were prepared having DXY/VE/BnCl(1:30) ratios of 2.5 ppm/15.6ppm (Example 33), 5 ppm/15.6 ppm (Example 34), 1 ppm/31.2 ppm (Example35), and 2.5 ppm/31.2 ppm (Example 36), respectively. FIG. 25 is a bargraph showing that each hydrogel achieved 100% killing efficiency. FIG.26 is an isobologram demonstrating the synergy of a doxycycline/cationicpolymer against P. aeruginosa when delivered by loaded hydrogels Example33 and Example 35, indicated by a drug combination dose to the left ofthe line of additivity, represented by a square inside the triangle. Theleft square corresponds to Example 35, the right square to Example 33.The drug combination is effective at extremely low doxycyclineconcentration (about 1 ppm, or 1 mg/L).

Biofilm Eradication by ABA Triblock/Cationic Polymer Hydrogels

The cationic polymer loaded hydrogels were investigated for theirability to eradicate biofilms. The formation of biofilms occurs asmicrobes adheres to a surface (inanimate material or tissue) and as theyproliferate, they can secrete insoluble gelatinous exopolymers thatresults in a three-dimensional cell:polymer matrix known as a biofilm.Manifestation of medical biofilm can be extremely challenging asmicrobes growing in biofilms are recalcitrant and drastically lessresponsive to antimicrobial agents and host defense systems compared tothe planktonic cells. Biofilm persistence can led to clinical conditionssuch as impaired wound healing, chronic inflammation and the spread ofinfectious emboli.

To establish relevance to antimicrobial agents used in biofilmelimination, various microbes (S. aureus, E. coli and C. albicans) werecultured for several days for the development of biofilm prior to thetreatment. Cationic polymer VE/BnCl(1:30) or cationic polymerVE/PrBr(1:30) was loaded at MBC concentration for S. aureus, E. coli orC. albicans into VitE1.25-PEG(20k)-VitE1.25 (4 wt. %) hydrogels andplaced onto the biofilm. Cationic polymer loaded hydrogels Example 37and Example 38 were prepared for S. aureus, cationic polymer loadedhydrogels Example 39 and Example 40 were prepared for E. coli, andcationic polymer loaded hydrogels Example 41 and Example 42 wereprepared for C. albicans. The corresponding blank hydrogel Example 15was used as a control. The culture was then incubated with the hydrogelsfor 24 hours for antimicrobial actions to occur. XTT assay was thenperformed to evaluate the viability of the remaining microbe. In thisassay, a lower optical density (O.D.) reading corresponds to lower cellviability and better biofilm elimination capacities of the hydrogels.

FIGS. 27A and 27B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of S. aureus biofilms by blankhydrogel Example 15 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25),cationic polymer loaded hydrogel Example 37 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30)), and cationicpolymer loaded hydrogel Example 38 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30)). A reductionin metabolic activity (FIG. 27A) and biomass (FIG. 27B) was obtainedwith the cationic polymer loaded hydrogels against S. aureus.

FIGS. 28A and 28B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of E. coli biofilms by blankhydrogel Example 15 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25),cationic polymer loaded hydrogel Example 37 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30)), and cationicpolymer loaded hydrogel Example 38 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30)). A reductionin metabolic activity (FIG. 28A) and biomass (FIG. 28B) was obtainedwith the cationic polymer loaded hydrogels against E. coli.

FIGS. 29A and 29B are bar graphs showing the reduction in metabolicactivity and biomass, respectively, of C. albicans biofilms by blankhydrogel Example 15 (containing 4 wt. % VitE1.25-PEG(20k)-VitE1.25),cationic polymer loaded hydrogel Example 37 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0156 wt. % VE/BnCl(1:30)), and cationicpolymer loaded hydrogel Example 38 (containing 4 wt. %VitE1.25-PEG(20k)-VitE1.25 and 0.0625 wt. % VE/PrBr(1:30)). A reductionin metabolic activity (FIG. 29A and biomass (FIG. 29B) was obtained withcationic polymer loaded hydrogels against C. albicans.

It can be seen that hydrogels loaded with VE/BnCl(1:30) were asefficient as those loaded with VE/PrBr(1:30) in reducing theproliferation and viability of S. aureus (Gram-positive) and C. albicans(fungus). The major difference was observed in E. coli (Gram-negative)where cell treated with hydrogels loaded with the more hydrophobicVE/BnCl(1:30) had significantly lower viability compared to thosetreated with VE/PrBr(1:30)-loaded hydrogels.

The persistence of microbial biomass after treatment was quantifiedusing the crystal violet assay. Any portion of the biofilm that remainscan act as a dormant zone harboring pockets of microbes that stayprotected from antimicrobial agents. Furthermore, there can also besubpopulations of resistant cells referred to as ‘persisters’ that mayreside in the residual biofilm. The ability of the cationic polymerloaded hydrogels to eradicate biomass followed a trend similar to thetrend in reduction of cell viability of the microbes residing in thebiofilms. That is, VE/BnCl(1:30) had better antimicrobial action thanVE/PrBr(1:30) against E. coli, and the two cationic polymers displayedsimilar efficiency against S. aureus and C. albicans biofilms.

SEM imaging demonstrates that biofilms treated with VE/BnCl(1:30)-loadedhydrogel (Example 37) showed extensive cell destruction and clearance(FIG. 30). Only ruptured cell fragments with the absence of intact cellsremain for S. aureus and C. albicans. The images also correlate well tothe quantification assays of cell viability and biomass whereVE/BnCl(1:30) hydrogel is significantly more effective in eradicating E.coli biofilm compared to the VE/PrBr(1:30) counterpart.

Preparation and Testing of PEG-Diamine Based Block Copolymers

The term “PEG-diamines” is a general term that encompasses the morespecific polymers PEG10DA and PEG20DA.

Nuclear Magnetic Resonance (NMR) Spectroscopy. The ¹H NMR spectra ofmonomers and polymers were recorded on a Bruker Advance 400 NMRspectrometer at 400 MHz at room temperature. The ¹H NMR measurementparameters: acquisition time of 3.2 seconds, pulse repetition time of2.0 seconds, 30° C. pulse width, 5208-Hz spectral width, and 32 K datapoints. Chemical shifts were referred to the solvent peak (6=7.26 forCDCl₃).

Gel permeation chromatography (GPC). GPC analysis for polymers wascarried out on a GPC system (Waters 2690, MA, U.S.A.) with an OptilabrEX differential refractometer detector (Wyatt Technology Corporation,U.S.A.) and Waters HR-4E column. The mobile phase used was THF with aflow rate of 1 mL/min. Number-average molecular weights andpolydispersity indices were calculated from a calibration curve obtainedusing a series of polystyrene standards (Polymer Laboratories Inc., MA,U.S.A., with molecular weight ranging from 1,350 to 151,700).

Rheological experiments. The hydrogels were prepared by dissolving thepolymers (4.0 wt %, 5.0 wt %, 6.0 wt % and 8.0 wt %) in DI water at 25°C. The rheological analysis of the hydrogels was performed on an ARES-G2rheometer (TA Instruments, U.S.A.) equipped with a plate-plate geometryof 8 mm diameter. Measurements were taken by equilibrating the gels at25° C. between the plates at a gap of 1.0 mm. The data were collectedunder a controlled strain of 3.0% and a frequency scan of 1.0 to 100rad/second. Gelation properties of the polymer solutions was monitoredby measuring the shear storage modulus (G′), as well as the loss modulus(G″), at each point. To investigate the shear-thinning properties, theviscosity of the hydrogels was monitored as a function of shear ratefrom 0.1 to 10 sec⁻¹.

The PEG-diamine-based block copolymers were prepared according to thefollowing general reaction scheme using PEG10DA or PEG20DA.

The degree of polymerization (DP) of the central poly(ethylene oxide)block is designated by the subscript n, where n has an average value ofabout 250 (corresponding to the DP of PEG10DA) or 500 (corresponding tothe DP of PEG20DA). The DP of each peripheral polycarbonate block isdesignated by the subscript m, which can have an average value of about1 to about 10. The degree of conjugation of the cyclic carbonate monomerafter the ROP is designated by the value 2×m.

Example 45

Preparation of VitE1.05-PEG10DA-VitE1.05, m=1.05, n=250. The followingprocedure is representative. PEG10DA (M_(n) 10,790 Da, 0.216 g, 0.02mmol) and MTC-VitE (0.115 g, 0.2 mmol) were added to a small vessel andmixed evenly. The vessel was connected to an oil pump and the reactionmixture was stirred slowly under high vacuum for 10 minutes. Then, thevessel was heated to 120° C. with stirring. Heating was continued for 36hours, at which time the vessel was cooled to room temperature. Theresulting mixture was dissolved in dichloromethane (DCM, 2 mL). Theresulting solution was poured into diethyl ether (40 mL) to precipitatethe product. The precipitate was centrifuged and washed three times withdiethyl ether. Finally, the wet solid was dried in vacuo, givingVit1.05-PEG10DA-VitE1.05 as a white powder (0.21 g, 94%). PDI, 1.10. ¹HNMR (400 MHz, CDCl₃): δ 4.58 (dd, 8H, MTC—CH₂—), 3.64 (s, 909H, H ofPEG), 2.60 (s, 4H, Ph-CH₂CH₂— of VitE), 1.70-2.25 (m, br, 23H, Ph-CH₃and Ph-CH₂CH₂— of VitE), 1.00-1.70 (m, 57H, H of VitE and MTC-CH₃), 0.87(t, 25H, 4× —CH₃ on the hydrophobic tail of VitE).

Example 46

Preparation of VitE1.8-PEG20DA-VitE1.8, m=1.8, n=500.VitE1.8-PEG20DA-VitE1.8 was prepared according to the general procedureof Example 45 using PEG20DA. Yield, 90%. PDI, 1.08. ¹H NMR (400 MHz,CDCl₃): δ 4.49 (s, br, 14H, MTC—CH₂—), 3.64 (s, 1818H, H of PEG), 2.56(s, 7H, Ph-CH₂CH₂— of VitE), 1.70-2.12 (m, 40H, Ph-CH₃ and Ph-CH₂CH₂— ofVitE), 1.00-1.60 (m, 97H, H of VitE and MTC-CH₃), 0.87 (t, 43H, 4× —CH₃on the hydrophobic tail of VitE).

Example 47

Preparation of VitD1.0-PEG10DA-VitD1.0, m=1.0, n=250.VitD1.0-PEG10DA-VitD1.0 was prepared according to the general procedureof Example 45 using PEG10DA and MTC-VitD2. Yield, 93%. PDI, 1.09. ¹H NMR(400 MHz, CDCl₃): (4.30 (m, 8H, MTC—CH₂—), 3.65 (s, 909H, H of PEG),1.10 2.90 (m, 48H, H of VitD and MTC-CH₃), 0.75-1.10 (m, 24H, VitD-CH₃),0.54 (s, 6H, Vit-CH₃).

Example 48

Preparation of VitD1.3-PEG20DA-VitD1.3, m=1.3, n=500.VitD1.3-PEG20DA-VitD1.3 was prepared according to the general procedureof Example 45 using PEG20DA and MTC-VitD2. Yield, 91%. PDI, 1.08. ¹H NMR(400 MHz, CDCl₃): (4.29 (m, br, 10H, MTC—CH₂—), 3.65 (s, 1818H, H ofPEG), 1.10 2.90 (m, 62H, H of VitD and MTC-CH₃), 0.75-1.10 (m, 31H,VitD-CH₃), 0.54 (s, 8H, Vit-CH₃).

Example 49

Preparation of VitE1.15-PEG10DA-VitE1.15, m=1.15, n=250 using a ROPcatalyst (LiCl) and a solvent (NMP). PEG10DA, MTC-VitE and LiCl (molarratio of the feed is 1:10:0.05) were dissolved in a small volume ofN-methyl pyrrolidone (NMP) and heated for 24 hours at 70° C. and cooledto room temperature. The product was precipitated in diethyl ether,centrifuged and washed with diethyl ether for 3 times, and dried invacuo, yielding VitE1.15-PEG10K-VitE1.15 as a white powder (yield 88%,PDI 1.13). ¹H NMR (400 MHz, CDCl₃): δ 4.48 (dd, 9H, MTC—CH₂—), 3.64 (s,909H, H of PEG), 2.60 (s, 5H, Ph-CH₂CH₂— of VitE), 1.65-2.25 (m, br,25H, Ph-CH₃ an Ph-CH₂CH₂— of VitE), 1.00-1.60 (m, 62H, H of VitE andMTC-CH₃), 0.87 (t, 28H, 4× —CH₃ on the hydrophobic tail of VitE).

Example 50

Preparation of VitE0.97-PEG20DA-VitE0.97, m=0.97, n=250VitE0.97-PEG20K-VitE0.97 was prepared according to the general procedureof Example 49 using PEG20DA. Yield, 90%. PDI, 1.11. ¹H NMR (400 MHz,CDCl₃): δ 4.47 (s, br, 8H, MTC—CH₂—), 3.64 (s, 1818H, H of PEG), 2.57(s, 4H, Ph-CH₂CH₂— of VitE), 1.72-2.20 (m, 21H, Ph-CH₃ and Ph-CH₂CH₂— ofVitE), 1.00-1.60 (m, 52H, H of VitE and MTC-CH₃), 0.85 (t, 23H, 4× —CH₃on the hydrophobic tail of VitE).

Examples 51-67 were prepared according to the above-described generalprocedures using MTC-VitE and PEG10DA or PEG20DA, and varying solvent,catalyst, reaction time and temperature. The conditions and degree ofconjugation of the cyclic carbonate monomer are summarized in Table 13.

Table 13 summarizes the triblock copolymers of Examples 45-67.

TABLE 13 Molar ratio of PEG-diamine: Cyclic Degree of Ex- Polymer CyclicPEG- Monomer: Temp Duration Conjugation ample Name Monomer diamineCatalyst Comment (° C.) Solvent (h) (2 × m) 45 VitE1.05- MTC- PEG10DA1:10:0 in vacuo 120 no 36 2.06 PEG10D VitE solvent A- VitE1.05 46VitE1.8- MTC- PEG20DA 1:10:0 in vacuo 120 no 36 3.59 PEG20D solventA-VitE VitE1.8 47 VitD1.0- MTC- PEG10DA 1:10:0 in vacuo 120 no 36 2.02PEG10D VitD2 solvent A- VitD1.0 48 VitD1.3- MTC- PEG20DA 1:10:0 in vacuo120 no 36 2.63 PEG20D VitD2 solvent A- VitD1.3 49 VitE1.15- MTC- PEG10DA1:10:0.05 70 NMP 24 2.30 PEG10K- VitE (LiCl) VitE1.15 50 VitE0.97 MTC-PEG20DA 1:10:0.05 70 NMP 24 1.93 PEG20K- VitE (LiCl) VitE0.97 51 MTC-PEG10DA 1:10:0 r.t. DCM 16 1.02 VitE 52 MTC- PEG10DA 1:6:0 65 DMF 160.76 VitE 53 MTC- PEG10DA 1:10:3 (Et₃N) r.t. DMF 16 0.44 VitE 54 MTC-PEG10DA 1:10:3 (Et₃N) 65 DMF 16 0.76 VitE 55 MTC- PEG10DA 1:10:0 r.t.DCM 90 1.17 VitE 56 MTC- PEG10DA 1:10:0 65 CHCl₃ 45 0.12 VitE 57 MTC-PEG10DA 1:10:0 90 NMP 24 1.44 VitE 58 MTC- PEG10DA 1:10:0 N₂ 120 no 362.10 (low VitE atmosphere solvent yield) 59 MTC- PEG20DA 1:10:0 r.t. DCM16 0.63 VitE 60 MTC- PEG20DA 1:6:0 65 DMF 16 0.42 VitE 61 MTC- PEG20DA1:10:3 (Et₃N) r.t. DMF 16 0.29 VitE 62 MTC- PEG20DA 1:10:3 (Et₃N) 65 DMF16 0.37 VitE 63 MTC- PEG20DA 1:10:0 r.t. DCM 90 0.96 VitE 64 MTC-PEG20DA 1:10:0 65 CHCl₃ 45 0.034 VitE 65 MTC- PEG20DA 1:10:0 N₂ 120 no36 1.91 (low VitE atmosphere solvent yield) 66 MTC- PEG20DA 1:10:0 70NMP 24 1.41 VitE 67 MTC- PEG20DA 1:10:0 90 NMP 24 1.24 VitE

The Table 13 results indicate the use of organic solvent in the reactionled to an incomplete conjugation, and the catalyst-free enabled ease ofpurification and recycling of excess MTC-VitE and MTC-VitD2 monomers.

FIG. 31 (graph) shows the GPC diagrams of Vit1.05-PEG10DA-VitE1.05(Example 45) and VitE1.8-PEG20DA-VitE1.8 (Example 46). The elutioncurves showed single unimodal peaks. No oligomer peaks were observed,indicating that no back-biting reactions took place under theseconditions. Also, the block copolymers were found to be nearlymonodisperse with a polydispersity of 1.10 and 1.08, respectively.

The compositions of the triblock copolymers were estimated from 1H NMRspectroscopy by quantitative comparisons between integral intensities ofthe peak of methyl protons of VitE or VitD2 and those of the ethylenegroups of the central poly(ethylene oxide) block. FIG. 32 is a protonNMR spectrum of Example 45, Vit1.05-PEG10DA-VitE1.05, in CDCl3. Themolar ratio of MTC-VitE to PEG10DA was determined to be 2.10 bycomparison of the integral intensities of the peaks of methyl groups ofthe VitE fragment at 0.87 ppm with ethylene protons of the centralpoly(ethylene oxide) block at 3.64 ppm.

Hydrogels of the PEG-diamine base triblock copolymers were then preparedby dissolving a given block polymer in HPLC grade water at 25° C. atvarious concentrations. Table 14 summarizes the G′ (storage modulus) andG″ (elastic modulus) values at a frequency of 1 Hz of hydrogels formedat 4, 5, 6, and 8 wt. % solids based on total weight of the hydrogel.

TABLE 14 Polymer Concentration Example Block Copolymer (wt %) G′ (Pa) G″(Pa) 45 VitE1.05-PEG10DA- 4 1,621 299 VitE1.05 5 5,064 742 6 9,287 1,2448 14,508 1,671 46 VitE1.8-PEG20DA- 4 561 85 VitE1.8 5 1,809 320 6 1,8661,401 8 13,703 1,189 47 VitD1.0-PEG10DA- 4 680 391 VitD1.0 5 1,694 1,0216 2,766 1,652 8 6,907 3,816 48 VitD1.3-PEG20DA- 4 2,611 890 VitD1.3 53,401 1,131 6 7,693 2,517 8 10,148 3,098

The polymer concentration significantly influenced the storage modulusG′ of the hydrogels. Increasing the concentration led to higher storagemoduli (G′), demonstrating that hydrogel stiffness can be easily tunedby varying polymer concentration. Also, as shown in FIG. 33 (graph), thehydrogels formed with Example 45 exhibited pronounced shear-thinningbehaviour, facilitating its ability for topical application.

In Vitro Release of Herceptin from PEG-Diamine Based Block Copolymers

Loaded hydrogels (0.25 mL) containing herceptin (10 g/L) were preparedat 4 wt. % polymer as described further above. The loaded hydrogels weretransferred to Transwell inserts (Corning, U.S.A.). No precipitation orleftover solution was seen after gelation, showing that all theHerceptin was loaded into the hydrogels. The inserts were then immersedin 25 mL of phosphate buffered saline (pH 7.4) release medium and wereshaken using a water bath at 100 rpm at 37° C. At designated timeintervals, 0.1 mL of the release medium was removed and replaced withfresh medium. The released amount of Herceptin was quantified using theprotein quantification BCA assay (Pierce, U.S.A.).

FIG. 34 (graph) shows that the release of Herceptin from a 4 wt. %hydrogel formed with Example 46 was sustained for 42 days withapproximately 60% of the antibody being released cumulatively. Therelease occurred in a biphasic manner where about 45% of the antibodywas released in the initial 15 days followed by a much slower release inthe later phase. These data showed that Herceptin release from the gelwas more sustained compared to block copolymers prepared with MTC-VitEusing PEG-diol (compare FIG. 4B with FIG. 34)

In Vivo Biocompatibility and Gel Degradation Studies

All animal experiments were conducted in accordance with the approvedprotocol from the Institutional Animal Care and Use Committee (IACUC) atthe Biological Resource Centre of Singapore. Female balb/c mice,weighing 20-25 g were injected with 150 μL of blank 4 wt. % hydrogelsubcutaneously. For histological examination, the hydrogel and itssurrounding tissue were isolated. At 1 day, 3 days, 1 week, 2 weeks, 4weeks and 6 weeks post injection, the hydrogel and its surroundingtissue was excised. The samples were fixed in 4% neutral bufferedformalin and then stained with hematoxylin-eosin (H&E) using standardtechniques. Optical photomicrographs of histological specimens wereexamined.

FIG. 35 is a set of photographs and staining images of the loadedhydrogel prepared with Example 46 in the mouse tissue at various timesup to 6 weeks. Within the initial first week post-injection, thehydrogel (bracketed regions) remained largely intact. At 2 weeks postinjection, there was reduction in gel thickness, which suggest that thehydrogel had begun to degrade. By 6 weeks, the hydrogel still remainedvisible during the excision procedure as well in H&E stained tissuesections, demonstrating that the degradation of this gel was slower thanthat of hydrogels prepared with MTC-VitE using PEG-diol.

CONCLUSIONS

Biodegradable and biocompatible vitamin E-functionalized “ABA”-typetriblock copolymers were formed by organocatalyzed ring opening ofcyclic carbonate monomers bearing a covalently bound form of a vitamin Ecompound. The block copolymers form physically cross-linked hydrogelsand organogels without the addition of reagents or chemical reactions.The rheological properties of the gels can be readily tunable in facilemanner by varying the polymer concentration or composition. Gelstiffness, indicated by the storage modulus G′, can vary from 1000 to12000 Pa depending on the polymer composition and/or concentration. Awide array of pharmaceutical compounds, including small molecule drugs,biomolecules and cosmetics/dietary products can be loaded into the gelsduring the gel formation process. These gels function as depots fordelivery of drugs having controlled release profiles. With the ease offormulation and tunability, these soft physical gels serve as anattractive candidate for an extensive range of pharmaceutical-drivenapplications.

As one example, a biocompatible and biodegradable herceptin loadedhydrogel prepared with VitE1.25-PEG(20k)-VitE1.25 was successfullyemployed as an injectable local delivery material for the antibody.Rheological properties and porosity of the loaded hydrogel can beadjusted by varying the polymer composition and polymer concentration.Histological examination reveals that the hydrogel does not inducechronic inflammatory response, and is able to degrade in vivo over time.The hydrogel matrix provides sustained release of herceptin, andlocalizes the antibody within the tumor site. In vivo anti-tumorefficacy is significantly enhanced using a single subcutaneous injectionof herceptin-loaded hydrogel at a site close to the tumor as compared toherceptin solution. Herceptin-loaded hydrogel injected once at a distalsite away from the tumor is comparable to that of weekly intravenousadministration of herceptin solution over 4 weeks. Herceptin treatmentusing the hydrogel requires less frequent injections, thereby providinggreater convenience and improved patient compliance. These resultssuggest that the vitamin E-functionalized hydrogels hold promise forsubcutaneous delivery of antibodies.

The cationic polymer loaded hydrogels are able to eradicate the biomassand greatly reduce viability of microbes residing in biofilms. Takentogether, the results suggest that the cationic polymer loaded hydrogelscan be used to eliminate both planktonic and biofilm microbes.

PEG-diamine based triblock copolymers, which have a carbamate linkinggroup joining the blocks, were synthesized by bulk ring openingpolymerization at elevated temperature without solvent or catalyst. Thecatalyst-free synthesis enables an easy purification step and recyclingof excess monomers. These triblock copolymers formed hydrogels at 4 wt.% that showed pronounced shear-thinning behaviour suitable for medicalinjections. The hydrogel stiffness can be easily tuned by varyingpolymer concentration. Release was more sustained and controlledrelative to PEG-diol based triblock copolymers, and the in vivo gellifetime was longer for the PEG-diamine based polymers. The hydrogelshave utility as injectable carriers for subcutaneous delivery ofantibodies and other therapeutic drugs, or dressings for wound healing.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. When a range is used to express apossible value using two numerical limits X and Y (e.g., a concentrationof X ppm to Y ppm), unless otherwise stated the value can be X, Y, orany number between X and Y.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiments were chosen and described in order to best explain theprinciples of the invention and their practical application, and toenable others of ordinary skill in the art to understand the invention.

What is claimed is:
 1. A block copolymer having the structure:

wherein each V′ is selected from the group consisting of

and combinations thereof, d′ is a positive number having an averagevalue of 100 to 600, and each m′ is an independent positive numberhaving an average value between 1 and
 2. 2. The block copolymer of claim1, wherein V′ is


3. The block copolymer of claim 2, wherein each m′ has a value in therange of 1.03 to 1.78.
 4. The block copolymer of claim 2, wherein eachm′ has a value in the range of 1.03 to 1.3.
 5. The block copolymer ofclaim 1, wherein d′ is about
 500. 6. The block copolymer of claim 1,wherein V′ is


7. The block copolymer of claim 6, wherein each m′ has a value in therange of 1.03 to 1.78.
 8. A drug composition, comprising: about 4 wt. %to about 10 wt. % of the block copolymer of claim 1; a solvent; andabout 0.0001 wt. % to about 10 wt. % of a drug; wherein weight percent(wt. %) is based on total weight of the drug composition, and the drugcomposition is a gel, wherein the block copolymer, the solvent, and thedrug are associated by noncovalent interactions.
 9. The drug compositionof claim 8, wherein V′ is


10. The drug composition of claim 8, wherein V′ is


11. The drug composition of claim 8, wherein the drug is an anti-tumoragent and the drug composition is suitable for treating a cancer. 12.The drug composition of claim 11, wherein the anti-tumor agent is amono-clonal antibody.
 13. The drug composition of claim 12, wherein themono-clonal antibody is herceptin.
 14. The drug composition of claim 8,wherein the solvent is water, the composition is a hydrogel, and thecomposition inhibits growth of cancer cells in vivo when administered bya depot injection.
 15. The drug composition of claim 8, wherein the drugis an antimicrobial cationic polycarbonate.
 16. The antimicrobial drugcomposition of claim 15, wherein the drug composition comprises a seconddrug that is an antimicrobial compound.
 17. The antimicrobial drugcomposition of claim 16, wherein the antimicrobial compound is selectedfrom the group consisting of fluconazole, doxycycline, and combinationsthereof.
 18. A method of treating a cancer, comprising performing an invivo depot injection of the drug composition of claim 8 near or incontact with a cancerous tumor.
 19. The method of claim 18, wherein thecancer is a breast cancer.
 20. A method of preparing a block copolymer,comprising heating under vacuum with agitation at a temperature of about50° C. to about 150° C. a mixture consisting of i) a cyclic carbonatemonomer comprising a pendent group containing a covalently bound form ofa vitamin and ii) a dinucleophilic polymeric initiator for a ringopening polymerization (ROP) of the cyclic carbonate monomer, thepolymeric initiator comprising a poly(ethylene oxide) chain and twoterminal *—NH₂ groups, thereby forming the block copolymer.